Use of bacteriocin-producing ethanologens in biofuel production

ABSTRACT

An ethanologen for producing biofuel from one or more carbohydrates and reducing lactate and acetate production in a biofuel manufacturing process. The ethanologen is made by introducing into the ethanologen one or more exogenous genes required for production of a bacteriocin. The resulting ethanologen reduces lactate and acetate production by contaminant lactic acid bacteria by expression of the bacteriocin during the biofuel manufacturing process. Certain resulting ethanologens ferment sugars not naturally or not preferentially utilized by  Saccharomyces cerevisiae  during the manufacturing process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional Application No.62/361,783 filed on Jul. 13, 2016, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 2011-67009-30043awarded by the USDA/NIFA. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This disclosure relates generally to biofuel production. Moreparticularly, this disclosure describes microbial ethanologensengineered to produce a bacteriocin, which inhibits contaminant lacticacid bacteria, thereby reducing lactate and acetate production duringbiofuel production.

BACKGROUND OF THE INVENTION

Microbial production of biofuels from carbohydrates (i.e., corn starch,cane sugar and lignocellulosic substrates) is a component of the UnitedStates' plan to reduce its dependency on fossil fuels. Themicroorganisms typically considered for the production of biofuelsinclude Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli,and Clostridium sp. However, all of these microorganisms suffer from oneor more of the following deficiencies: relatively low tolerance to theenvironmental stresses likely to be encountered in fermentation (e.g.,high levels of alcohols, acids, and/or osmolarity), complex physiology,poor availability of genetic tools, and limited ability to secreteenzymes.

Bacterial contaminants are a chronic problem in biofuel plants whichresult in significant financial losses to the biofuel industry. The mostcommon bacterial contaminants in ethanol plants are lactic acid bacteria(LAB). These organisms compete with yeast for nutrients, depletecarbohydrates that otherwise would have been converted into biofuel andproduce compounds inhibitory to the yeast (i.e., lactate and acetate).It is estimated that the financial loss related to the lactate/acetateproblem is approximately $500,000 to $800,000 USD for 50 million gallonsof ethanol per year per plant.

At the present time, the majority of the biofuels industry controlsthese bacterial contaminant issues through the use of antibiotics.However, the use of antibiotics is likely to decline significantly dueto concerns related to antibiotics used in biofuel plants entering thehuman food chain through dried distiller grains with soluble (DDGS), aby-product of the bioethanol industry which contributes 20% of therevenue to an ethanol plant. Major food companies have recognized thisconcern and publically-stated their intent to no longer source meat fromanimals that had been raised using DDGS containing antibiotics. A betterapproach to biofuel production might be to develop a LAB strain capableof outcompeting wild type LAB for nutrients while producing biofuelinstead of the deleterious lactate and acetate.

BRIEF SUMMARY OF THE INVENTION

The present invention is a genetically-engineered ethanologen capable ofproducing one or more bacteriocins, which are antimicrobial peptidesproduced by one type of bacteria to kill other bacteria, and,preferably, capable of fermenting sugars not naturally or notpreferentially utilized by Saccharomyces cerevisiae.

This improved ethanologen provides a means of enhancing ethanolproduction efficiency by reducing inefficiencies caused by bacterialcontamination in biofuel plants without the use of antibiotics, and byincreasing the total sugars that are converted to ethanol.

Accordingly, in a first aspect, the present invention provides anethanologen for inhibiting contaminant lactic acid bacteria present in abiofuel manufacturing process. Such an ethanologen includes anethanologen containing one or more exogenous genes required forproduction of a bacteriocin, whereby production of the bacteriocin bythe ethanologen inhibits contaminant lactic acid bacteria present in thebiofuel manufacturing process.

In certain embodiments, inhibition of the contaminant lactic acidbacteria results in reduced lactate and acetate levels in the biofuelmanufacturing process.

In certain embodiments, the ethanologen is capable of fermenting sugarsnot naturally or not preferentially utilized by a main fermentingmicrobe present in the biofuel manufacturing process, such asSaccharomyces cerevisiae.

In certain embodiments, the ethanologen is a native biofuel-producingorganism such as a Sacchoromyces sp. or Zymononas sp. In otherembodiments, the ethanologen is an organism engineered to produce abiofuel such as a lactic acid bacterium (i.e., Lactobacillus sp.,Lactococcus sp., Enterococcus, sp. or Streptococcus sp.), Escherichiasp., or Clostridium sp.

Exemplary biofuels encompassed by the present invention include ethanoland isobutanol.

Ethanologens according to the invention produce a Class I(lantibiotics), Class IIa (pediocins), Class IIb (two-peptide), or ClassIIc (cyclic) bacteriocins. Exemplary bacteriocins useful in theinvention include nisin, pediocin, brochocin-C, and carnocyclin A,respectively.

In certain embodiments, an ethanologen of the invention is aLactobacillus sp. engineered to produce the bacteriocin, preferably thespecies is Lactobacillus casei or Lactobacillus plantarum. In someembodiments, an ethanologen of the invention is a Lactococcus lactisengineered to produce the bacteriocin.

In some embodiments, the exogenous genes required for production of thebacteriocin are operably-linked to an inducible promoter.

Ethanologens of the invention are further envisioned, in certainembodiments, to include one or more immunity genes conferring resistanceto the ethanologen against the bacteriocin.

In a second aspect, the present invention provides abacteriocin-producing ethanologen for use in fermenting additionalsugars not naturally or not preferentially utilized by Saccharomycescerevisiae yeast. Exemplary ethanologens according to the invention areable to readily ferment pentose sugars or sugar polymers with a degreeof polymerization of 2, 3, 4, or more. Exemplary sugars not naturally ornot preferentially utilized by Saccharomyces cerevisiae yeast include,e.g., xylose, arabinose, trehalose, maltose, isomaltose, cellobiose,cellobiotriose, maltotriose, isomaltotriose, panose, raffinose,stachyose, maltotetraose, and maltodextrin.

In certain embodiments, the bacteriocin-producing ethanologen is anorganism engineered to produce a biofuel and bacteriocin lactic acidbacterium (i.e., Lactobacillus sp., Lactococcus sp., Enterococcus sp.,or Streptococcus sp.), Escherichia sp., or Clostridium sp.

In certain embodiments, the bacteriocin-producing ethanologen of theinvention is a lactic acid bacterium (i.e., Lactobacillus sp.,Lactococcus sp., Enterococcus sp., or Streptococcus sp.) engineered toco-utilize glucose and carbohydrates not naturally or not preferentiallyutilized by a primary fermentor microbe (e.g., Saccharomycescerevisiae), preferably the species being Lactobacillus casei orLactobacillus plantarum.

In some embodiments, the bacteriocin-producing ethanologen of theinvention is a Lactococcus lactis engineered to co-utilize glucose andcarbohydrates not naturally or not preferentially utilized bySaccharomyces cerevisiae yeast.

In some embodiments, the exogenous genes required for sugar utilizationare operably-linked to an inducible promoter.

Ethanologens according to the invention are useful in a broad range ofbiofuel manufacturing process, including those processes in which thecarbohydrate is provided in the form of a lignocellulosic feedstock.

In preferred embodiments, the biofuel manufacturing process is anantibiotic-free process.

In certain embodiments, the ethanologens are hop acid and/orantibiotic-resistant, thereby allowing for utilizing these antimicrobialingredients with the ethanologen

In certain embodiments, the inventive ethanologen is made by a methodincluding the steps of: (a) inactivating within a lactic acid bacteriumone or more endogenous genes encoding a lactate dehydrogenase ormannitol dehydrogenase; or (b) introducing into a lactic acid bacteriumone or more exogenous genes encoding a pyruvate decarboxylase and one ormore exogenous genes encoding an alcohol dehydrogenase II; (c)inactivating within a lactic acid bacterium one or more mannitoldehydrogenases; or (d) performing steps (a), (b) and (c) or acombination of any two steps (a), (b), and (c); and (e) introducing intothe lactic acid bacterium the one or more exogenous genes required forproduction of the bacteriocin; whereby the resulting engineeredbacterium produces significantly more ethanol than a wild-type lacticacid bacterium in an ethanol manufacturing process and reduces lactateand acetate production in the process by secretion of the bacteriocin,which inhibits contaminant lactic acid bacteria. The methodalternatively or in addition includes one or more steps of: (f)inactivating within a lactic acid bacterium genes encoding proteins suchas catabolite control protein A (ccpA) that are associated withcatabolite repression or elements related to catabolite repression; (g)removing DNA sequences such as catabolite responsive elements (cre) thatare involved in repressing the expression of genes required for uptakeand metabolism of sugars not naturally or not preferentially utilized bySaccharomyces cerevisiae yeast; (h) introducing into the lactic acidbacterium the one or more exogenous genes required for uptake andmetabolism of sugars not naturally or not preferentially utilized bySaccharomyces cerevisiae yeast; and (i) introducing into the lactic acidbacterium one or more genes for hop- and/or antibiotic-resistance.

The above method of making the inventive ethanologen may compriseinactivating within the lactic acid bacterium an endogenous geneencoding D-hydroxyisocaproate dehydrogenase. As well, the resultinglactic acid bacterium may include one or more of the following genedeletions: deletion (Δ) of the primary L-lactate dehydrogenase(ΔL-ldh1); Δ in one or more paralogs of L-lactate dehydrogenase(ΔL-ldh2, ΔL-ldh3, etcetera); Δ of D-lactate dehydrogenase (ΔD-ldh);and/or Δ of D-hydroxyisocaproate dehydrogenase (ΔD-hic). The ethanologenmay further include gene deletion mutation Δ mannitol dehydrogenase 1(Δmand1) and/or Δ mannitol dehydrogenase 2 (Δmand2).

In some embodiments, the exogenous gene encoding a pyruvatedecarboxylase comprises the gene of Zymomonas mobilis that encodes forpyruvate decarboxylase (Pdc), and the exogenous gene encoding an alcoholdehydrogenase II comprises the gene of Zymomonas mobilis that encodesfor dehydrogenase II (AdhII). Preferably, the exogenous genes aremodified to utilize codon usage preferences of the lactic acid bacteriumhost strain.

In preferred embodiments, the exogenous genes introduced in the lacticacid bacterium are operably linked to a promoter, such as a lactic acidbacterium such as phosphoglycerate mutase (pgm), as part of anexpression vector, such as pPpgm-PET. The promoter is preferably highlyexpressed in the stationary phase such as, for example, the GroELpromoter, the DnaK promoter, or the UspAC2 promoter.

A particularly useful ethanologen according to the invention is aLactobacillus casei 12A derivative with: (a) a deletion mutationΔL-ldh1, ΔL-ldh2, and ΔL-ldh3, an exogenous gene encoding a pyruvatedecarboxylase, and an exogenous gene encoding an alcohol dehydrogenaseII, wherein the exogenous genes are operably linked to a native L. caseipromoter, and wherein the engineered bacterium produces ethanol at agreater rate than a the wild-type Lactobacillus casei 12A bacterium; (b)one or more exogenous genes required for production of a bacteriocin,preferably pediocin, brochocin-C, nisin, or carnocyclin, whereby theetanologen is capable of fermenting sugars not naturally or notpreferentially utilized by a main fermenting microbe present in theethanol manufacturing process. The Lactobacillus casei 12A derivativeoptionally has one or more of: (c) a deletion mutation ΔccpA; (d) one ormore genes required for uptake and utilization of sugars not naturallyor not preferentially fermented by Saccharomyces cerevisiae yeast; and(e) one or more genes for hop- and/or antibiotic-resistance.

In another aspect, the invention encompasses a method of reducinglactate and acetate production in a biofuel manufacturing process. Sucha method includes steps of: (a) culturing an ethanologen according tothe invention on a substrate comprising a carbohydrate; (b) reducinglactate and acetate production by contaminant lactic acid bacteriapresent in the process by the ethanologen's secretion of a bacteriocin;and (c) collecting biofuel produced by the ethanologen. As can beappreciated, the present invention encompasses both the manufacture anduse of ethanolgens in reducing lactate and acetate production in abiofuel manufacturing process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Growth (▪,●) and glucose utilization (□,◯) by Lactobacilluscasei 12AΔ-ldh(pP_(PGM)-PET) (squares) and12AΔL-Idh1ΔL-Idh2ΔD-hic(pP_(PGM)-PET) (circles) at 37° C. in modifiedchemical defined media (mCDM; Diaz-Muñiz and Steele, 2006) containing10% glucose (w/v) with pH maintained at 6.0. See Diaz-Muñiz, I. and J.L. Steele, Antonie van Leeuwenhoek 90 (2006): 233-243.

FIG. 2A. Growth, glucose consumption, and ethanol production byLactobacillus casei 12AΔLldh1 (pP_(PGM)-PET).

FIG. 2B. 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP_(PGM)-PET) (2B) at 37° C. in achemically defined media containing 10% glucose with pH maintained at6.0.

FIG. 3. Metabolism of pyruvate (PYR) in Lactobacillus casei 12A andderivatives. The pyruvate related enzymes and pathways present in L.casei 12A: L-lactate dehydrogenases (L-Ldh); D-lactate dehydrogenase(D-Ldh); D-Hydroxyisocaproate dehydrogenase (D-Hic); acetolactatesynthase (Als); oxaloacetate decarboxylase (Oad); pyruvate kinase (Pyk);phophoenolpyruvate carboxikinase (Pck); pyruvate-formate lyase (Pfl);alcohol dehydrogenase (Adh). The enzymes and pathway from Zymomonasmobilis are shown as thick arrows: pyruvate decarboxylase (Pdc); alcoholdehydrogenase (Adh). Abbreviations: EMP, Embden-Meyerof-Parnas pathway;Glu, glucose; PEP, phosphoenolpyruvate.

FIG. 4. Schematic illustrating the gene replacement procedure developedfor gene replacement in L. casei 12A. Presence of the pheS* loci resultsin sensitivity to p-Cl-Phe. This phenotype (derivatives with pheS* formsmaller colonies) allows for selection derivatives that have undergonerecombination resulting in loss of the pheS* loci (derivatives withoutphe* form bigger colonies).

FIG. 5A. Construction of PET cassette in pTRKH2. PET cassette sequencewas obtained from Zymomonas mobilis. Codon usage of pdc and adhII wereoptimized specifically for L. casei 12A using Java Codon Adaptation Tool(Jcat). Codon optimized cassette was synthesized then cloned into pTRKH2for expression in L. casei 12A.

FIG. 6B. Detail of gene organization in the PET cassette: Ppgm, nativepromoter from L. casei 12A phosphoglycerate mutase; ribosomal bindingsite (RBS); pdc, pyruvate decarboxylase; adhII, alcohol dehydrogenase;Pin structure, native L. casei 12A transcriptional terminator of kdgRtranscriptional regulator protein. The cassette was flanked by PstI andBamHI restriction sites for cloning into pTRKH2.

FIG. 6. Growth curves of L. casei 12A and 12A Δldh1 transformed withempty pTRKH2 (control) or pPgm-PET growth in chemically defined medium(CDM) for 48 hrs. Working cultures were prepared from frozen stocks bytwo sequential transfers in MRS broth (see J. C. de Man, M. Rogosa andM. Elisabeth Sharpe, Appl. Bact. 23. 130-135 (1960)) with incubationsconducted statically at 37° C. for 24 hrs and 18 hrs, respectively.These cultures were then transferred to mCDM overnight and monitoredevery 6 hrs for OD600 (optical density at 600 nm).

FIG. 7. SDS-PAGE agar overlay bacteriocin inhibition assay usingconcentrated supernatants. Lane 1, 50 ng of pediocin standard; Lane 2,Lb. casei E2 (pNZ8048); Lane 3, Lb. casei E2 pNZPed+.

FIG. 8. Inducible GusA expression by L. casei in response to differentlevels of sakacin AIP.

FIG. 9. Physical map of gene clusters encoding bacteriocins used asexamples in this application. The fill patterns identify ORFs coding thebacteriocin structural gene (black), transport proteins (dark gray),modification or maturation (light gray), immunity (stippled), and otherfunctions (white). Map is not to scale.

FIG. 10. Changes in pH after 72 h in model ethanol fermentations withLb. casei E3 (pNZPed+; “Bac Positive”), the non-bacteriocin producingLb. casei E3 control (“Bac Negative”), and the yield reducing bacteriumLb. plantarum LS1.

FIG. 11. Lactate levels (w/v) after 72 h in model ethanol fermentationsusing Lb. casei E3 (pNZPed+; “Bac Positive”), the non-bacteriocinproducing Lb. casei E3 control (“Bac Negative”), and the yield reducingbacterium Lb. plantarum LS1.

FIG. 12. Final levels (w/v) of DP1 and DP2 carbohydrates after 72 h inmodel ethanol fermentations using Lb. casei E3 (pNZPed+; “BacPositive”), the non-bacteriocin producing Lb. casei E3 control (“BacNegative”), and the yield reducing bacterium Lb. plantarum LS1.

FIG. 13. Ethanol yields (percent, w/v) after 72 h in model ethanolfermentations using Lb. casei E3 (pNZPed+; “Bac Positive”), thenon-bacteriocin producing Lb. casei E3 control (“Bac Negative”), and theyield reducing bacterium Lb. plantarum LS1.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby any later-filed non-provisional applications.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising”,“including”, and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are nowdescribed. All publications and patents specifically mentioned hereinare incorporated by reference for all purposes including describing anddisclosing the chemicals, instruments, statistical analysis andmethodologies which are reported in the publications which might be usedin connection with the invention. All references cited in thisspecification are to be taken as indicative of the level of skill in theart. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

II. The Invention

The present invention is directed to improving the production efficiencyand capacity of bioenergy production from carbohydrates by developing anantibiotic-free process to control bacterial infections in thefermentation and which is also able to convert a greater percentage ofthe available carbohydrates to biofuel. Bioenergy production viafermentation of carbohydrate is increasing worldwide, and microbialcontamination has a strong negative effect on process efficiency andprofitability. A variety of microorganisms contaminate bioethanolfermentations, however, lactic acid bacteria (LAB), and particularlylactobacilli, are of primary concern. Losses caused by microbialcontamination at individual US bioethanol plants, for example, have beenestimated at $14.5 million per year, and there are over 200 bioethanolplants operating in the US. Use of antibiotics, primarily penicillin andvirginiamycin, is currently the most common means for control, but thispractice has led to the emergence of antibiotic-resistant contaminants,and it may also affect the value of fermentation byproducts such asdried distillers grains with solubles. Thus, research is needed togenerate more competitive and innovative solutions for USagriculture-based bioenergy systems.

It is the inventors' hypothesis that representative strains of the mostcommon bacterial contaminants can be metabolically engineered to thwartchronic and acute infections in bioenergy plants, reducing currentreliance on antibiotics and increasing bioenergy product yields.Specifically, the inventors hypothesize that ethanologens can beengineered to: i) produce the bioenergy molecule of interest; ii)produce bacteriocins with a broad spectrum of inhibition; iii) be highlyresistant to hop acids and antibiotics; and iv) convert to ethanolcarbohydrates that are not naturally or not preferentially fermented bythe yeast. Used like “starter cultures” for food fermentations, theseorganisms would be inoculated into the fermentor at the start of eachrun. The engineered ethanologens will grow to high numbers in thefermentation vessel, killing “wild” bacteria and converting sugarspreviously used by bacterial contaminants into ethanol rather thanlactic or acetic acids. They will also produce ethanol from simple andcomplex sugars that are not naturally or not preferentially used byyeast. To test the hypothesis, the inventors have used a Lactobacilluscasei ethanologen they have constructed as a model to demonstrateinhibition—via heterologous expression of different bacteriocins—ofbacterial contaminants recovered from bioethanol facilities.

Microbial contamination in bioenergy production. Saccharomycescerevisiae is the biocatalyst of choice in the commercial production ofbio-based chemicals such as ethanol and lactate. These fermentations arenot conducted under sterile conditions, and selection of a eukaryoticbiocatalyst such as S. cerevisiae enables the use of antibiotics tocontrol bacterial infections that can impede processing efficiency. Forexample, it is estimated that approximately 85% of fermentationfacilities in the United States producing ethanol from corn starchutilize antibiotics to control bacterial infections. The production ofbiofuels (both ethanol and isobutanol) from lignocellulosic feedstocksare likely to suffer from even greater difficulties with bacterialinfections, as the hurdles to bacterial growth (osmotic pressure andbiofuel concentration) are lower in these fermentations. However, thereis a growing consensus in the industry that use of antibiotics tocontrol bacterial contamination in commercial biofuel fermentations is aproblem, due to the size of the industry and the use of large quantitiesof a biofuel fermentation by-product, dried distillers grains withsolubles (DDGS), as animal feed. Consumer concerns regarding antibioticsin foods has led numerous large food companies (i.e., McDonalds,Walmart, Tyson Foods, Chipotle) to announce they will soon halt thepurchase of meat from animals that have consumed or have been treatedwith antibiotics. An antibiotic-free solution to bacterial contaminationof commercial fermentation facilities is urgently needed.

A variety of microorganisms contaminate bioethanol fermentations,however, lactic acid bacteria (LAB), and particularly lactobacilli, areof primary concern. Contamination by lactobacilli results in losses tothe bioethanol industry due to both chronic and acute infections.Chronic infections are expected and tolerated, although theysignificantly reduce productivity, due in large part to the lactobacillicompeting with the yeast for carbon and other nutrients. Nonetheless,these losses are substantial with estimates of approximately 0.1 to 1%(w/v) ethanol productivity loss when lactobacilli reach 10⁷ organismsper ml of mash, and higher levels are commonly observed in the industry.Acute infections occur when lactobacilli reach high numbers early in thefermentation, resulting in sufficient lactic acid being produced toinhibit the yeast and cause a “stuck” fermentation. When this occurs,the fermentation facility must be shut down and cleaned. Acuteinfections have become less common in recent years, but remain asignificant financial burden to the ethanol industry. Thus, processimprovements that effectively control bacterial contamination withoutuse of antibiotics would substantially improve the production efficiencyand capacity of the US agriculture-based bioenergy industry. Suchtechnology would carry the added social benefit of reducing theincidence of antibiotics in human and animal food.

The predominance of LAB contaminants in commercial production ofbio-based materials by yeast reflects the fact that yeast and LABco-inhabit the same environments. Both are commonly found innutritionally rich environments where their evolutionary strategyinvolves rapid conversion of carbohydrates into metabolic end productsthat inhibit competitors. This relationship has been harnessed by humansin the production of a wide array of products including sour doughbread, kefir, sour beers, wine, fish sauce, and soy sauce. Theirlong-term coexistence likely has resulted in yeasts evolving to behighly resistant to low pH and high levels of organic acids, while LABhave evolved to be among the most alcohol tolerant bacteria known.

This disclosure describes an approach to controlling LAB contaminationof industrial yeast bioenergy fermentations that leverages the naturalecology of yeast and LAB. In brief, the inventors use L. casei as amodel to demonstrate that ethanologens can be engineered to displacecontaminating bacteria while simultaneously producing the desiredbioenergy molecule from carbon sources that were formerly used bycontaminants as well as residual sugars that are not naturally or notpreferentially fermented by the biocatalytic yeast. Thus, in addition tocontrolling LAB contamination, the inventors' approach enhances theproduction efficiency of bioenergy from fermentation of agriculturalfeedstocks.

High level innate resistance to many environmental stressors. Microbialbiocatalysts often must be highly resistant to a variety ofenvironmental stressors, including elevated concentrations of desiredend products. Resistance to inhibitors in lignocellulosic substrates andbiofuel products, for example, is one of the most challenging obstaclesin the development of an efficient biocatalyst for alcohol production byfermentation [37-41]. The robust nature of L. casei and otherlactobacilli is evidenced by the ability of these species to grow inhigh acid, high salt, cold and warm environments with or without oxygen[42]. Moreover, lactobacilli are among the most innately alcoholtolerant (ethanol and isobutanol) microorganisms characterized to date,including S. cerevisiae, Escherichia coli and Clostridium sp. [37] [40,43, 44]. This attribute is illustrated by the fact that a L. casei12A-derived ethanologen 12AE1, shows superior growth in modifiedsynthetic corn stover (mSynH) containing 3% ethanol as compared toZymomonas mobilis ATCC 31821 and an E. coli ethanologen, GLBRC E1(Vinay-Lara et al. 2016). Additionally, L. casei 12AE1 shows higherinnate resistance to osmotic, acetic acid, and lignotoxin stressors inmSynH than Z. mobilis ATCC 31821 or E. coli GLBRC E1. Furthermore,lactobacilli possess inducible general and specific mechanisms forenvironmental stress adaptation that can be manipulated to furtherincrease resistance to adverse conditions [17, 20, 26, 45-54].Collectively, existing knowledge of molecular mechanisms for innate andinducible stress tolerance provides a solid foundation for strainengineering strategies to maximize the fitness of Lactobacillus-basedplatforms.

Ability to utilize lignocellulosic-derived carbohydrates. An intrinsicability to utilize particular substrates is an important considerationin the selection of a biocatalyst. Co-fermentation of cellobiose andxylose, for example, is particularly important to biofuels fermentationbecause it supports simultaneous saccharification and fermentation oflignocellulosic feedstocks [55]. L. casei and L. plantarum possess genesfor utilization of many different sugars, including xylose, cellobiose,and other sugars present in lignocellulosic feedstocks [11,56]. Theinventors have engineered L. casei 12A to co-ferment xylose and glucose.These results illustrate how the rich diversity of carbohydrateutilization genes in lactobacilli can be leveraged to engineer strainssuited to a particular biocatalytic process.

Ability to produce and secrete heterologous enzymes. Somebio-manufacturing processes require enzyme pretreatments that addconsiderable cost to the method. Conversion of lignocellulose intofermentable substrate for biofuels manufacture, for example, is anenzyme intensive process, and lactobacilli such as L. casei and L.plantarum naturally produces enzymes such as f-glucosidases that couldreduce the need for added enzymes. These enzymes are not normallysecreted, but lactobacilli have an established record as a host forexport of native and heterologous proteins [57-65]. All Lactobacillusgenomes have been found to carry single copies of SecA, SecE, SecY, YajCand SecG, as well as two copies of YidC [66]. Mathiesen et al. [62] haveidentified signal sequences for efficient secretion of heterologousproteins by L. plantarum, which should function in other Lactobacillusspecies [66]. Additionally, lactobacilli possess several differentbinding mechanisms for surface associated proteins, including N- orC-terminally anchored proteins, lipoproteins, and LP×TG-anchoredproteins [66], which can be exploited for surface localization ofheterologous proteins [67-69]. This knowledge provides the foundationneeded to engineer Lactobacillus-based biocatalysts able to accommodatesimultaneous saccharification and bioproducts manufacture.

Bacteriocins. “Bacteriocins,” are, as used herein, defined asribosomally synthesised, proteinaceous substances that inhibit thegrowth of other bacteria, typically closely related species. Theseantimicrobial peptides are produced by many bacterial species, includingLAB. The bacteriocins produced by LAB have been studied in great depthdue to their potential for controlling spoilage and pathogenicmicroorganisms in foods. These molecules can be divided into two majorclasses, based on their structural and physical properties. Class Ibacteriocins are termed lantibiotics, and include small (<5 kDa),heat-stable peptides that are post-translationally modified toincorporate unusual amino acids such as lanthionine and/ormethyllanthionine. Class II bacteriocins are also small (<10 kDa),heat-stable peptides, but these molecules may or may not undergopost-translational modification. Class II bacteriocins are furtherseparated into Class IIa (pediocins and pediocin-like peptides), ClassIIb (two-peptide), or Class IIc (cyclic) bacteriocins. Both Class I andClass II bacteriocins include peptides that are active as singlepeptides as well as two-peptide systems where one or both of themolecules is inactive in single form. Subclasses of Class I and IIbacteriocins have also been established based on differences instructure or activity.

Like conventional antibiotics, bacteriocins may exhert bacteriostatic orbactericidal effects on susceptible cells. Conventional antibiotics canbe grouped into five major categories according to their target, andbacteriocins from LAB act against at least two of these pathways:disruption of the cell wall synthesis and disruption of the cytoplasmicmembrane. The well-characterized Class I lantibiotic nisin, for example,uses lipid II as a docking molecule, and also interacts with the lipidintermediates III and IV. Binding inhibits peptidoglycan synthesis, andtriggers subsequent formation of membrane pores that rapidly kill targetbacteria. Other lantibiotics (termed “type B”) also disrupt cell wallsynthesis by binding lipid II but do not form pores.

In contrast, several Class II bacteriocins target membrane components ofthe mannose-phosphotransferase (man-PTS) system, which somehow triggerspermeabilization of the cell membrane and rapid dissipation of themembrane potential. The variations that are observed in host specificityamong Class II bacteriocins are proposed to be due to the fact thatindividual bacteriocins recognize a limited number of the man-PTSs foundin different strains or species of bacteria. Finally, some Class IIbacteriocins that inhibit cells by disrupting the cytoplasmic membranedo not always target lipid II or the man-PTS. For example, therespective targets for two-peptide bacteriocins plantaricin JK andlactococcin G are an APC superfamily transporter and an enzyme involvedin peptidoglycan synthesis, undecaprenyl pyrophosphate phosphatase.Other Class II bacteriocins, including lacticin Q, enterocin AS-48, andcarnocyclin A, appear to interact directly with the lipid bilayer.

Spectrum of inhibition. Both within and across different classes,bacteriocins may display narrow or broad range of target species. Theinventors' analysis of the microbiological profile in commercial ethanolplants reveal a diversity of LAB species may be present, so successfulinhibition of this complex community requires the construction of L.casei strains able to produce bacteriocins with a broad spectrum ofactivity. Nisin, a lantibiotic produced by some strains of Lactococcuslactis, and Class II bacteriocins such as pediocin and brochocin C,which are made by strains of pediococci, lactobacilli, and Brochothrixcampestris, each display broad inhibitory activity against a variety ofLAB and other Gram-positive bacteria and have been studied for theirefficacy in controlling spoilage microbes in alcoholic fermentations.That examples presented herein demonstrate that addition of nisin tobeer fermentation inhibited spoilage bacteria but not brewing yeasts,and a pediocin-producing Lactobacillus sp. also inhibited beer spoilagebacteria. The inventors have successfully expressed synthetic geneclusters for pediocin A and brochocin C in an L. casei ethanologen, andconfirmed the recombinant strain is able to inhibit many of the spoilagebacteria we have collected from commercial ethanol facilities. Thepresent invention encompasses other bacteriocins that: i) show a broadspectrum of activity against Gram-positive bacteria; and/or ii) displaydifferent modes of action. The candidates described in the examplessection include the lantibiotic nisin, as well as carnocyclin A, a ClassIIc cyclic bacteriocin produced by Carnobacterium maltaromaticum.

As an exemplary embodiment, the inventors have developed a bioengineeredbiofuel-producing strain of Lactobacillus casei. The followingcharacteristics illustrate why L. casei and other lactobacilli are anideal biofuels fermentation organism: ability to uselignocellulosic-derived mono- and di-saccharides; resistance toenvironmental stresses likely to be encountered in industrial biofuelsfermentations, including high levels of biofuels, acids, and/orosmolarity; relatively simple fermentative metabolism with almostcomplete separation of cellular processes for biosynthesis and energymetabolism; possibility to direct metabolic flux of both pentoses andhexoses to pyruvate (allowing for construction of derivatives producingsecond generation biofuels (i.e., isobutanol); the availability ofestablished platforms for introducing and expressing foreign DNA;availability of a deep portfolio of molecular-genetic data related totheir ecological adaptation, genomics, transcriptomics, lipidomics, andmetabolomics; the ability to secrete and display proteins, hencepotential for use in consolidated bioprocessing; and designation as aGRAS (Generally Regarded As Safe) species.

L. casei 12A, a strain isolated from corn silage on the University ofWisconsin-Madison campus, was selected as the biofuels-producingparental strain, due to its alcohol resistance, carbohydrate utilizationprofile, and amenability to genetic manipulation.

A multi-pronged approach has been employed to redirect metabolic flux inL. casei 12A to ethanol. The first approach was to inactivate genes thatencode enzymes which compete with the 12A pathway to ethanol. The secondapproach utilized the introduction of synthetic genes modeled fromZymomonas mobilis that encode pyruvate decarboxylase (Pdc) and alcoholdehydrogenase II (Adh2) activities (PET cassette). These synthetic geneswere designed utilizing the L. casei codon usage for highly expressedgenes with a constitutive L. casei promoter (phosphoglycerate mutase),synthesized, ligated with digested pTRKH2 to form pP_(PGM)-PET), andintroduced into 12A derivatives by electroporation. This two prongedapproach has resulted in an L. casei 12A derivative that producesethanol as more than 80% of its metabolic end products.

The constructed derivative of L. casei 12A described in para. [0061]produces ethanol as more than 80% of its final metabolic end productsfrom glucose. This has been increased to greater than 90% by theinactivation of the two mannitol dehydrogenase genes and theintroduction of a second copy of the PET cassette under the control ofthe UspAC2 promoter. This is by far the greatest conversion that hasbeen reported with a lactobacilli, and will allow us to exploit theadvantages of the use of lactobacilli as biocatalysts for the productionof biofuels. These advantages are further delineated below.

The present invention further combines converting Lactobacillus casei12A to an ethanologen with the ability to express bacteriocins in lacticacid bacteria (LAB), including Lb. casei 12A. The bacteriocins producedby this approach allow the ethanologens to inhibit contaminating lacticacid bacteria (LAB) present in ethanol production processes, therebyreducing the side production of lactate/acetate and enhancing ethanolyield.

The specific features and advantages of the present invention willbecome apparent after a review of the following experimental examples.However, the invention is not limited to the specific embodimentsdisclosed herein.

III. EXAMPLES Example A

This example addresses: (1) what level of carbohydrate Lactobacilluscasei 12A derivatives are capable of using; and (2) what level ofethanol production takes place at elevated glucose concentrations.

In the first experiment, 48 small volume (2 ml) fermentations wereconducted in GC vials containing our L. casei chemically defined mediato examine glucose utilization and end product formation. In parallel,these fermentations were conducted in a 96 well plate reader to monitorgrowth. The experimental matrix was: 3 levels of glucose (2.5, 5.0, and10% w/v), with and without the osmoprotectants present in ACSH (0.7 mMbetaine, 0.7 mM choline chloride, and 0.2 mMDL-carnitine), with andwithout 2.5 μg/ml erythromycin (Ery) to select for the plasmid encodedPET cassette, and four different strains. The strains utilized were: (1)an L. casei 12A derivative (12AΔL-ldh1) lacking L-lactate dehydrogenase1 (L-ldh1), the primary fermentative lactate dehydrogenase, with pTRKH2(empty vector control); (2) 12AΔL-ldh1 containing pPPGMPET, pTRKH2 withan insert containing the L. casei codon optimized Zymomonas mobilisgenes encoding pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II(Adh2) activities under the control of the L. casei phosphoglyceratemutase (pgm) promoter, (3) an L. casei 12 A derivative(12AΔL-ldh1ΔL-ldh2ΔD-hic) lacking L-ldh1, L-ldh2, andD-hydroxyisocaproate dehydrogenase (D-Hic) containing pTRKH2; and (4)12AΔL-ldh1ΔL-ldh2ΔD-hic containing pPPGM-PET. These fermentations wereconducted at 37° C. for 96 h and the media had an initial pH of 6.0.Three of the strains (12AΔL-ldh1(pTRKH2), 12AΔL-ldh(pP_(PGM)-PET) and12AΔL-ldh1ΔLldh2ΔD-hic (pP_(PGM)-PET) reached an OD600 of greater than1.0 within 24 h and grew at indistinguishable rates regardless of theglucose concentration, the presence or absence of eitherosmoprotectants, or Ery. The other strain, 12AΔL-ldh1ΔL-ldh2ΔD-hic(pTRKH2) grew poorly, never reaching an OD600 of greater than 0.05, evenafter 96 h, regardless of media composition; this corresponds withprevious experiments and was expected, as this strain lacks an efficientmechanism to regenerate NAD+ from pyruvate.

The addition of osmoprotectants did not have a significant effect ongrowth of any of the strains under the conditions examined; however, thepresence of the osmoprotectants did result in a reduction in lysis ofstrains producing ethanol in the presence of 2.5% glucose. No lysis wasobserved by the ethanol producing strains at the higher glucoseconcentrations, suggesting that the higher osmolarities induced genesthat provide enhanced ethanol tolerance. The most significant findingfrom the growth experiments is that growth of L. casei 12A derivativesis not affected by the glucose (osmolarity) concentrations up to 10%,rather, these conditions seem to enhance cell viability in stationaryphase of 12A derivatives producing ethanol.

Metabolic end product accumulation in the small volume fermentationswere determined by GLBRC Enabling Technologies (HPLC-RID), and theresults for L. casei 12AΔL-ldh (pP_(PGM)PET) and 12AΔL-ldh1ΔL-ldh2ΔD-hic(pP_(PGM)-PET) are presented in Table 1. All of the glucose was consumedin fermentations containing 2.5% (139 mM) and 5.0% (278 mM) glucose. Infermentations containing 10% (566 mM) glucose, glucose utilizationranged from 8.1 to 9.5% (459.1 to 536.4 mM). The ethanol formed in the2.5% (139 mM) glucose fermentations ranged from 1.3 to 1.4% (219.6 to247.6 mM), with % theoretical yields ranging from 79 to 89%. The ethanolformed in the 5.0% (278 mM) glucose fermentations ranged from 2.6 to2.7% (438.0 to 466.0 mM), with % theoretical yields ranging from 79 to84%. The ethanol formed in the 10% (566 mM) glucose fermentations rangedfrom 3.3 to 3.8% (563 to 651.5 mM), with % theoretical yields rangingfrom 50 to 58%. In fermentations containing 10% (556 mM) glucose,significant accumulation of mannitol (73.2 to 92.4 mM) was observed,suggesting that pyruvate decarboxylase activity has become limiting.Under all the conditions examined, L. casei 12AΔLldh1ΔL-ldh2ΔD-hic(pP_(PGM)-PET) produced slightly more ethanol and slightly less lactatethan L. casei 12AΔl-ldh (pP_(PGM)-PET). Possible reasons for incompleteglucose utilization in fermentations containing 10% glucose includechanges in the pH of the media and increases in pressure due toconducting the fermentations in closed vials. To overcome these issues,fermentations that allow for pH control and CO₂ release have beenconducted.

Fermentations with 10% glucose with osmoprotectants and Ery have beenconducted in our larger scale (500 ml) fermentation equipment thatallows for pH control and CO₂ release with L. casei 12AΔL-ldh(pP_(PGM)-PET) and 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP_(PGM)-PET) at 37° C.,with pH maintained at 6.0. The growth and glucose utilization (enzymaticdetermination) results are presented in FIG. 1. Growth of the twostrains are indistinguishable under these conditions; however, greaterglucose utilization was observed by 12AΔL-ldh (pP_(PGM)-PET).

The 19 12A derivatives that were constructed via our two-step genereplacement method are presented in Table 2, clearly demonstrating thesuccessful construction of a variety of 12A mutants.

TABLE 1 Metabolic end products accumulated by L. casei 12A ethanologensduring growth in a chemically defined medium (initial pH 6.0) containinga different levels of glucose, with and without osmoprotectants at 37°C. for 96 hrs. Osmo- Glucose (mM) Products (mM) Final % Ethanol Strainprotectant Int Con Rem EtOH Man Lac Ace pH (v/v)* 12A ΔL-Ldh1 − 142.0142.0 BQL 227.9 BQL 12.3 6.6 5.4 1.3 (pP_(PGM)- PET) 12A ΔL-Ldh1 − 277.0277.0 BQL 438.0 14.1 26.6 4.9 4.7 2.6 (pP_(PGM)- PET) 12A ΔL-Ldh1 −499.7 393.7 106.0 600.0 73.2 31.8 5.3 4.5 3.5 (pP_(PGM)- PET) 12AΔL-Ldh1 + 137.3 137.3 BQL 219.6 BQL 11.8 6.6 6.2 1.3 (pP_(PGM)- PET) 12AΔL-Ldh1 + 278.1 278.1 BQL 445.4  3.3 39.1 3.2 4.7 2.6 (pP_(PGM)- PET)12A ΔL-Ldh1 + 499.3 469.6  29.6 563.0 86.1 40.5 5.3 4.4 3.3 (pP_(PGM)-PET) 12A ΔL-Ldh1/ − 142.3 142.3 BQL 247.6 BQL 7.7 8.7 7.4 1.4ΔL-Ldh2/ΔD-Hic (pP_(PGM)- PET) 12A ΔL-Ldh1/ − 282.6 282.6 BQL 443.5 18.317.9 9.1 7.2 2.6 ΔL-Ldh2/ΔD-Hic (pP_(PGM)- PET) 12A ΔL-Ldh1/ − 508.0401.1 106.9 625.0 91.6 22.3 11.7 6.6 3.6 ΔL-Ldh2/ΔD-Hic (pP_(PGM)- PET)12A ΔL-Ldh1/ + 139.5 139.5 BQL 233.2 BQL 7.6 8.0 6.1 1.4 ΔL-Ldh2/ΔD-Hic(pP_(PGM)- PET) 12A ΔL-Ldh1/ + 280.9 280.9 BQL 466.0 18.1 15.2 8.3 6.72.7 ΔL-Ldh2/ΔD-Hic (pP_(PGM)- PET) 12A ΔL-Ldh1/ + 507.9 408.9  99.0651.5 92.4 26.0 12.4 6.8 3.8 ΔL-Ldh2/ΔD-Hic (pP_(PGM)- PET)Abbreviations: BQL = Below Quantitative Level. Abbr: Int—initial,Con—consumed, Rem—remaining, EtOH—ethanol, Man = Mannitol, Lac—Lactate,Ace—acetate.

TABLE 2 Lactobacillus casei 12A derivatives constructed in the past 10months in the Steele laboratory via gene replacement. Single knockoutsDouble knockouts Triple knockouts Quadruple knockouts ΔL-ldh1*ΔL-ldh1/ΔL-ldh2* ΔL-ldh1/ΔL-ldh2/ΔL-ldh3 ΔoadA/Δpck/Δpyc/Δfum ΔL-ldh2*ΔL-ldh1/ΔL-ldh3 ΔL-ldh1/ΔL-ldh2/ΔL-ldh4 ΔoadA/Δpck/Δpyc/Δaspal ΔL-ldh3ΔL-ldh1/ΔL-ldh4 ΔL-ldh1/ΔLldh2/ΔD-ldh ΔL-ldh4 ΔL-ldh1/ΔD-ldhΔL-ldh1/ΔL-ldh2/ΔD-hic* ΔD-ldh ΔL-ldh1/ΔD-hic ΔoadA/Δpck/Δpyc ΔD-hicΔL-ldh1/Δpck Δals ΔoadA/Δpck Δald ΔoadA/Δpyc Δa/drc Δpyc/Δpck ΔoadA ΔpycΔpck Δaspal Abbreviations: L-ldh-L-lactate dehydrogenase, D-ldhD-lactate dehydrogenase, D-hic-D-hydroxyisocaproate dehydrogenase,als-acetolactate synthase, ald-alpha acetolactate decarboxylase,a/drc-acetoin/diacetyl reductase, oad-oxaloacetatedecarboxylase,pyc-pyruvate carboxylase,pck-phosphoenolpyruvatecarboxikinase, aspal-aspartate-ammonia lyase, fum-fumarase.Asterisk-derivatives transformed with pP_(PGM)-PET are available.

Example B

This example describes analysis of the data the inventors obtained fromthe fermentations with 10% glucose with osmoprotectants and Ery thatwere conducted in our larger scale (500 ml) fermentation equipment withLactobacillus casei 12AΔL-ldh (pP_(PGM)-PET) and 12AΔL-ldh1ΔLldh2ΔD-hic(pP_(PGM)-PET) at 37° C., with pH maintained at 6.0. The inventors couldaccommodate three fermentation vessels at a time. Therefore, only the12AΔL-ldh (pP_(PGM)-PET) fermentation was conducted in duplicate.

The growth, glucose utilization, and ethanol production shown by thesestrains are presented in FIG. 2A (L. casei 12AΔL-ldh (pP_(PGM)-PET)) and2B (L. casei 12AΔL-ldh1ΔLldh2ΔD-hic (pP_(PGM)-PET)). The growth of thetwo strains under these conditions was indistinguishable. However,12AΔL-ldh (pP_(PGM)-PET) utilized a greater quantity of glucose andproduced more ethanol than 12AΔL-ldh1ΔL-ldh2ΔD-hic (pP_(PGM)-PET). Theglucose utilization and ethanol formation obtained with 12AΔL-ldh(pP_(PGM)-PET) in the larger fermentation vessels was significantlygreater than that obtained in the small volume fermentations describedin Example A. The mostly likely reason for this difference is that thelarger vessels allow for pH control.

The metabolic end products formed and glucose utilized as a function oftime for these fermentations is presented in Tables 3 and 4. 12AΔL-ldh1(pP_(PGM)-PET) will be the focus of this discussion, due to its higherproductivity. This 12A derivative utilized 504.5 mM glucose (9.1%)glucose in 96 h and produced 934.7 mM of “pyruvate-derived” metabolicend products, which is 87.4% of the theoretical yield from glucose.Ethanol was produced at a level of 771.3 mM (4.5%), which was 82.5% ofthe metabolic end-products.

The second most abundant metabolic end product was mannitol, which waspresent at 110.1 mM after 96 h. Mannitol accumulation began atapproximately 21 h, at the same time, ethanol as a percentage of thetotal metabolic end products began to decrease (% ethanol in total),suggesting that pyruvate decarboxylase activity becomes limiting at thattime. This corresponds to the entry of this organism into stationaryphase, suggesting that the L. casei phosphoglycerate mutase (pgm)promoter used to drive expression of the PET cassette is poorlyexpressed in stationary phase. Mannitol accumulation was overcome byinactivation of the two mannitol dehydrogenase genes and theintroduction of a second copy of the PET cassette under the control ofthe UspAC2 promoter. This derivative is designated Lb. casei E3.

It is difficult to directly compare our results to what is knownconcerning other biocatalysts, due to differences in media andfermentation equipment utilized. However, the results obtained in theseL. casei 12AΔL-ldh1 (pP_(PGM)-PET) fermentations are most similar to theEscherichia coli GLBRCE1 synthetic hydrolysate fermentations reported bySchwalbach et al. (2012, AEM 78:3442) in E. coli. GLBRCE1 converted 338mM glucose into 477 mM ethanol, an ethanol yield of 70.5% of thetheoretical maximum. L. casei 12AΔL-ldh1 (pP_(PGM)-PET) converted 504.5mM glucose into 771.3 mM ethanol, an ethanol yield of 76.4% of thetheoretical maximum.

TABLE 3 Metabolic end products formed and glucose consumption byLactobacillus casei 12A ΔL-Ldh1 (pP_(PGM)-PET) at 37° C. in a chemicallydefined media containing 10% glucose with pH maintained at 6.0. % % TimeGlucose (mM) Products (mM) % Ethanol Ethanol Ethanol:Lactate (hr) RemCon Total EtOH Man Lac Ace yield in total (v/v) ratio (mM:mM) 0 534.9BQL 12.5 11.1 0.0 0.1 1.2 1.2 89.2 0.1 85 1 540.6 BQL 22.7 12.0 0.0 5.45.3 2.1 52.7 0.1 2 2 545.2 BQL 24.0 12.8 0.0 5.5 5.7 2.2 53.4 0.1 2 3544.8 BQL 19.2 12.8 0.0 2.5 3.8 1.8 67.0 0.1 5 4 536.5 BQL 21.5 15.0 0.02.3 4.2 2.0 70.0 0.1 7 5 531.4 BQL 33.5 27.1 0.0 1.3 5.0 3.1 81.0 0.2 206 544.4 BQL 28.2 21.7 0.0 0.9 5.6 2.6 77.0 0.1 25 7 542.2 BQL 36.8 29.40.0 1.8 5.7 3.4 79.8 0.2 17 8 551.2 BQL 42.3 34.4 0.0 2.1 5.8 4.0 81.30.2 16 9 539.9 BQL 51.2 42.8 0.0 2.7 5.8 4.8 83.5 0.2 16 10 535.1 BQL67.0 58.1 0.0 3.3 5.6 6.3 86.7 0.3 18 11 513.9 21.0 74.9 66.1 0.0 3.65.2 7.0 88.2 0.4 18 12 521.1 13.8 90.6 82.5 0.0 2.9 5.2 8.5 91.0 0.5 2813 512.8 22.1 105.1 96.8 0.0 3.4 4.9 9.8 92.1 0.6 29 14 506.3 28.7 124.5116.1 0.0 3.9 4.4 11.6 93.3 0.7 29 15 490.8 44.1 140.0 131.7 0.0 4.4 4.013.1 94.0 0.8 30 16 481.5 53.5 158.7 150.1 0.0 4.9 3.7 14.8 94.6 0.9 3117 463.4 71.5 175.4 166.6 0.0 5.4 3.4 16.4 95.0 1.0 31 18 439.5 95.4196.7 187.6 0.0 5.9 3.2 18.4 95.4 1.1 32 19 442.9 92.0 223.0 213.2 0.06.7 3.1 20.8 95.6 1.2 32 20 419.4 115.5 235.4 225.5 0.0 7.0 2.8 22.095.8 1.3 32 21 414.8 120.1 258.5 247.6 0.4 7.8 2.7 24.2 95.8 1.4 32 22402.2 132.7 275.1 263.0 1.1 8.4 2.7 25.7 95.6 1.5 31 23 389.1 145.8292.6 278.7 2.1 9.2 2.5 27.3 95.3 1.6 30 24 412.0 122.9 297.6 281.5 3.49.8 2.8 27.8 94.6 1.6 29 25 375.9 159.0 336.1 318.4 4.4 10.8 2.5 31.494.7 1.9 29 26 357.3 177.6 353.2 332.4 5.7 11.8 3.3 33.0 94.1 1.9 28 27343.8 191.1 363.0 339.7 7.8 12.4 3.1 33.9 93.6 2.0 27 28 339.7 195.2387.7 362.0 9.4 13.3 2.9 36.2 93.4 2.1 27 29 336.6 198.3 411.0 382.910.5 14.3 3.2 38.4 93.2 2.2 27 30 318.4 216.5 411.7 383.1 11.1 14.7 2.938.5 93.0 2.2 26 32 292.4 242.5 451.8 421.5 12.9 15.1 2.3 42.2 93.3 2.528 34 289.7 245.2 481.8 445.1 16.3 17.4 2.9 45.0 92.4 2.6 26 44 221.6313.3 588.7 533.1 29.3 22.8 3.4 55.0 90.6 3.1 23 50 187.2 347.7 656.5584.7 41.6 25.7 4.4 61.4 89.1 3.4 23 58 151.4 383.5 714.0 623.4 56.328.5 5.8 66.7 87.3 3.6 22 66 118.6 416.3 768.6 664.1 65.3 31.5 7.7 71.886.4 3.9 21 70 99.9 435.0 813.2 691.8 78.8 33.4 9.2 76.0 85.1 4.0 21 7492.2 442.7 827.3 702.8 81.3 33.8 9.4 77.3 85.0 4.1 21 82 62.4 472.5873.8 744.0 81.1 36.8 11.9 81.7 85.1 4.3 20 90 44.1 490.8 913.8 764.497.8 38.3 13.2 85.4 83.7 4.5 20 96 30.4 504.5 934.7 771.3 110.1 39.214.1 87.4 82.5 4.5 20

TABLE 4 Metabolic end products formed and glucose consumption byLactobacillus casei 12A ΔL-Ldh1/ΔL-Ldh2/ΔD-Hic (pP_(PGM)-PET) at 37° C.in a chemically defined media containing 10% glucose with pH maintainedat 6.0. % Ethanol % Time Glucose (mM) Products (mM) % in total EthanolEthanol:Lactate (hr) Rem Con Total EtOH Man Lac Ace yield product (v/v)ratio (mM:mM) 0 575.3 BQL 13.3 13.3 0.0 0.0 0.0 1.2 100.0 0.1 — 1 557.118.3 12.9 12.9 0.0 0.0 0.0 1.1 100.0 0.1 — 2 553.1 22.3 12.7 12.5 0.00.0 0.2 1.1 98.1 0.1 — 3 566.0 9.3 15.2 14.5 0.0 0.0 0.6 1.3 96.0 0.1 —4 541.8 33.6 16.7 15.2 0.0 0.0 1.5 1.5 90.9 0.1 — 5 548.8 26.5 21.7 18.70.0 0.0 3.0 1.9 86.2 0.1 — 6 543.2 32.1 25.8 21.0 0.0 0.0 4.9 2.2 81.20.1 — 7 551.2 24.1 31.6 25.5 0.0 0.0 6.1 2.7 80.6 0.1 — 8 554.9 20.436.8 30.6 0.0 0.0 6.2 3.2 83.1 0.2 — 9 551.3 24.0 45.6 38.5 0.0 1.1 6.04.0 84.4 0.2 36 10 532.7 42.6 55.5 48.7 0.0 0.9 5.9 4.8 87.6 0.3 53 11532.3 43.0 64.5 57.7 0.0 1.1 5.8 5.6 89.4 0.3 54 12 544.0 31.3 76.6 70.60.0 0.0 6.1 6.7 92.1 0.4 — 13 536.6 38.7 87.7 82.1 0.0 0.0 5.6 7.6 93.60.5 — 14 519.6 55.8 101.5 93.7 0.0 2.6 5.2 8.8 92.3 0.5 36 15 510.2 65.1111.9 107.0 0.0 0.0 4.9 9.7 95.7 0.6 — 16 513.9 61.4 134.2 125.5 0.0 3.84.8 11.7 93.6 0.7 33 17 474.2 101.1 134.7 130.7 0.0 0.0 3.9 11.7 97.10.8 — 18 485.3 90.0 167.7 158.3 0.0 5.5 3.9 14.6 94.4 0.9 29 19 463.7111.6 179.9 170.4 0.0 5.9 3.5 15.6 94.8 1.0 29 20 462.1 113.2 199.4189.4 0.0 6.8 3.2 17.3 95.0 1.1 28 21 459.0 116.3 218.4 207.8 0.0 7.63.0 19.0 95.1 1.2 27 22 451.9 123.5 236.9 224.9 0.7 8.6 2.8 20.6 94.91.3 26 23 426.3 149.0 238.0 235.8 0.0 0.0 2.3 20.7 99.0 1.4 — 24 380.6194.7 314.8 299.9 3.5 9.4 1.9 27.4 95.3 1.8 32 25 426.0 149.3 295.3279.1 3.6 10.4 2.2 25.7 94.5 1.6 27 26 384.5 190.8 297.1 283.3 2.5 9.61.7 25.8 95.3 1.7 29 27 365.0 210.3 301.5 289.6 0.5 9.6 1.8 26.2 96.11.7 30 28 371.3 204.1 331.6 317.2 1.7 11.0 1.6 28.8 95.7 1.9 29 29 360.5214.8 339.2 320.3 5.6 10.9 2.3 29.5 94.4 1.9 29 30 332.9 242.4 341.2326.4 2.9 10.9 1.0 29.7 95.7 1.9 30 32 333.5 241.8 395.1 375.6 5.8 12.51.2 34.3 95.1 2.2 30 34 296.7 278.6 381.7 363.9 5.3 12.1 0.4 33.2 95.32.1 30 44 272.5 302.8 520.6 480.5 22.4 14.4 3.2 45.2 92.3 2.8 33 50245.1 330.2 578.6 527.3 30.5 16.6 4.2 50.3 91.1 3.1 32 58 216.0 359.4627.0 564.1 39.4 17.6 5.8 54.5 90.0 3.3 32 66 194.1 381.3 681.0 606.249.2 18.3 7.2 59.2 89.0 3.5 33 70 175.2 400.1 700.5 618.2 54.8 19.5 8.060.9 88.3 3.6 32 74 169.5 405.8 706.8 622.6 56.7 19.2 8.3 61.4 88.1 3.632 82 149.8 425.5 712.2 639.6 63.1 0.0 9.5 61.9 89.8 3.7 — 90 141.0434.3 752.9 672.0 70.1 0.0 10.8 65.4 89.3 3.9 — 96 126.6 448.7 774.2663.1 79.0 21.1 10.7 67.3 85.7 3.9 31Glucose))×100% Ethanol=(mmol/L ethanol×46.068 g/mol)/(1000 mg/g)×(1000ml/L/100 ml)×(0.789 g/ml).

Example C

Screening Strains of L. casei for Biofuels' Relevant Phenotypes andGenes.

Our laboratory has a culture collection which contains approximately 60strains of L. casei isolated from green plant material (i.e., cornsilage), cheese, wine, and humans. The eleven strains with genomesequences were screened for the ability to utilize 60 differentcarbohydrates, including numerous carbohydrates present inlignocellulosic feed stocks. Individual strains were able to grow onbetween 17 and 26 different substrates. The strains isolated from cornsilage (12A and 32G) grew on the greatest number of substrates. Ninegene clusters potentially involved in cellobiose utilization and onegene cluster involved in xylose utilization were identified.

The eleven strains with genomic information were also screened foralcohol tolerance (ethanol, 1-propanol, 1-butanol, and2-methyl-1-butanol), growth in AFEX-pretreated corn stover hydrolysate(ACSH), and transformation (electroporation) efficiency. L. casei 12Aexhibited the greatest tolerance to the biofuels examined. For example,when grown in the presence of 10% ethanol, it reached a final celldensity 40% of that it attained in the absence of ethanol. Of the 11strains examined for growth in corn stover hydrolysate, 3 of thesestrains (ATCC 334, 21-1, and 12A) grew significantly better, reaching afinal optical density at 600 nm of approximately 2.0 within 28 h. FiveL. casei strains were examined for transformation efficiency with pTRKH2(O'Sullivan and Klaenhammer 1993). L. casei 12A exhibited a frequency(approximately 5×10⁵ transformants per ug of pTRKH2) at least 50-foldhigher than that observed with any of the other strains examined. Basedupon the results from these analyses, L. casei 12A was selected as thebiofuel producing parental strain.

Completing the L. casei 12A Genome.

For further information regarding the L. casei 12A genome, seeBroadbent, et al., BMC Genomics 2012, 13:533, which is incorporated byreference herein. To enhance the depth of genomic sequence coverage of12A, genomic DNA was prepared and submitted to the Joint GenomeInstitute (JGI) for genome sequencing. A draft genome of L. casei 12Awith approximately 500× coverage assembled into 397 scaffolds wasreceived from JGI. This genome assembly was subsequently merged with theprevious 23×454-generated paired end genome assembly in collaborationwith personnel from DuPont Inc. (Madison, Wis.), yielding a genomeassembly with 19 ordered contigs. We have generated PCR amplicons acrossall 19 gaps, and have sequenced 10 of these amplicons.

L. casei Metabolic Models.

We have developed a genome-scale metabolic model for L. casei ATCC334(the neotype strain) and 12A using the ModelSEED database and the genomeannotation from RAST. We have modified the draft L. casei 12A model fromModelSEED using the following processes: 1) thermodynamically infeasiblecycles were removed, 2) elementally imbalanced metabolic reactions werecorrected; and 3) model predictions for amino acid requirements werecompared against experimental growth phenotypes determined in alactobacilli chemically defined medium (CDM) described by Christensenand Steele (J. Bacteriol. 185 (2003): 3297-3306). Inconsistencies werecorrected by the addition or deletion of some reactions.

Redirecting Metabolic Flux in L. casei 12A to Ethanol.

The development of a method to inactivate genes in L. casei was arequirement for the construction of a L. casei strain capable ofconverting lignocellulosic biomass to ethanol. An efficient genereplacement method based on the introduction of pCJK47-based constructs(Kristich et al. 2007) via a 12A optimized electroporation protocol wasdeveloped.

A multi-pronged approach was employed to redirect metabolic flux in L.casei 12A to ethanol. The first approach is to inactivate genes thatencode enzymes which compete with the 12A pathway to ethanol, which hasacetyl-CoA as an intermediate. There are a large number of genes thatencode enzymes potentially involved in anaerobic pyruvate metabolism inL. casei. We have inactivated 9 of these genes: pyruvate-formate lyase(Pfl), the four L-lactate dehydrogenases (L-ldh1, Lldh2, L-ldh3, andL-ldh4), D-lactate dehydrogenase (D-ldh), D-hydroxyisocaproatedehydrogenase (DHic), acetolactate synthase (Als), and oxaloacetatedecarboxylase (OadA). Additionally, 5 derivatives lacking two or threeof the dehydrogenases have been constructed. Characterization of the endproduct distribution these mutants is presented in Table 5. The highestlevel of metabolic redirection to ethanol achieved to date using thisapproach, is 21%, achieved with the 12A ΔL-ldh1ΔL-ldh2ΔD-hic derivative.This derivative was determine to accumulate mannitol under highcarbohydrate loading. Mannitol accumulation was overcome by inactivationof the two mannitol dehydrogenase genes and the introduction of a secondcopy of the PET cassette under the control of the UspAC2 promoter. Thisderivative is designated Lb. casei E3.

The second approach utilized to direct metabolic flux in 12A towardsethanol was the introduction of synthetic genes modeled from Zymomonasmobilis that encode pyruvate decarboxylase (Pdc) and alcoholdehydrogenase II (Adh2) activities (PET cassette). These genes weredesigned utilizing the L. casei codon usage for highly expressed geneswith a constitutive L. casei promoter (phosphoglycerate mutase),synthesized by GeneArt, ligated with digested pTRKH2 (pPGM-PET), andintroduced into 12A derivatives by electroporation. Characterization ofthe end product distribution of two of these derivatives has beencompleted and is presented in Table 5. The highest level of metabolicredirection to ethanol achieved to date using this approach is 85.3%,achieved with the 12A ΔL-ldh1ΔL-ldh2 (pP_(PGM)-PET) derivative. It isinteresting to note that 12A derivatives with pP_(PGM)-PET grow morerapidly than their corresponding strains, suggesting that ethanol isless inhibitory to 12A derivatives than lactate.

These results suggest that the multi-pronged approach is effective forredirecting 12A metabolic flux to ethanol.

TABLE 5 Growth, substrate consumption, and metabolic end products formedby Lactobacillus casei 12A and derivatives during growth in a chemicallydefined media at 37° C. for 48 hrs. Concentration (mM) Growth SubstrateMetabolic End Products EtOH/ Max T_(d) Utilization^(a) (% of total)^(b)Yield Lac Derivative OD₆₀₀ (h.) Glc Cit Total L-lac D-lac EtOH Ace Man(%)^(c) ratio^(d) 12A 1.05 8.1 51.5 0.6 112.2 105.4, (94)  3.3, (3) 1.4,(1) 2.1, (2) BQL 108 0.0 12A ΔL-ldh1 1.28 7.0 52.6 11.5 87.0 42.3, (49)28.2, (32) 16.5, (19) BQL BQL 68 0.2 12A ΔL-ldh2 1.01 8.3 53.1 5.8 111.8105.0, (94)  3.2, (3) 1.6, (1) 2.0, (2) BQL 95 0.0 12A ΔL-ldh3 1.02 8.052.7 9.0 110.2 103.0, (94)  3.2, (3) 2.1, (2) 1.9, (2) BQL 90 0.0 12AΔD-ldh 1.02 7.7 51.9 BQL 112.4 103.5, (92)  5.4, (5) 1.1, (1) 2.4, (2)BQL 108 0.0 12A ΔD-hic 1.26 7.9 51.5 BQL 112.0 109.7, (98)  BQL 0.5, (1)1.8, (2) BQL 109 0.0 12A ΔL-ldh1/ 1.26 7.1 52.8 15.0 86.7 32.1, (37)42.6, (49) 12.0, (14) BQL BQL 64 0.2 ΔD-ldh 12A ΔL-ldh1/ 1.11 9.7 51.213.9 79.8 64.9, (81) BQL 14.9, (19) BQL BQL 61 0.2 ΔD-hic 12A ΔL-ldh1/0.93 9.4 52.5 10.3 71.4 BQL 51.5, (72) 18.6, (26) BQL 1.3, (2) 57 0.4ΔL-ldh2/ΔD-ldh 12A ΔL-ldh/ 0.52 31.3 21.7 12.8 36.1 0.6, (2) 0.4, (1) 7.6, (21)  7.2, (20) 20.3, (56) 52 7.6 ΔL-ldh2/ΔD-hic 12A (pTRKH2) 1.0113.2 52.5 BQL 108.9 100.4, (92)  7.6, (7) BQL 0.9, (1) BQL 104 0.0 12A(pPGM-PET) 0.95 6.79 51.3 8.2 95.1 14.8, (16) 13.2, (14) 58.1, (61) 9.0, (10) BQL 80 2.1 12A ΔL-ldh1 1.11 11.3 52.3 2.7 87.7 41.6, (47)34.0, (39) 12.1, (14) BQL BQL 80 0.2 (pTRKH2) 12A ΔL-ldh1 1.03 6.8 51.016.3 102.1 2.7, (3) 5.0, (5) 84.5, (83)  9.8, (10) BQL 76 10.9(pPGM-PET) 12A ΔL-ldh1/ 1.01 7.9 50.9 16.0 100.2 0.7, (1) 5.1, (5) 85.3,(85) 9.1, (9) BQL 75 14.7 ΔL-ldh2 (pPGM-PET) ^(a)Reported by the initialconcentration of glucose or citrate subtracted by the fetalconcentration of the respective compound at 48 hrs. ^(b)In parenthesis,metabolic end product distribution by % of total. ^(c)Calculated bypercentage of total metabolic end products produced/2 × (glucose +citrate) in mmoles. ^(d)Expressed as molar ratio, where lactate is thesummation of both the L- and D- forms. Abbreviations: BQL = belowquantifiable level; NA = not applicable; Glu = glucose; Cit = citrate;Lac = lactate; ETOH = ethanol; Ace = acetate; Man = mannitol

Example D. Conversion of a Lactic Acid Bacterium Lactobacillus casei 12Ato an Ethanologen

Lactobacillus casei 12A was selected as the biofuels parental strainbased upon its alcohol tolerance (grows in the presence of >10%ethanol), carbohydrate utilization, and relatively high transformationefficiency. This organism metabolizes hexoses through theEmbden-Meyerhof-Parnas pathway and converts pyruvate to lactate via avariety of different enzymes; including four L-lactate dehydrogenases(Ldh), one D-Ldh, and one D-hydroxyisocaproate dehydrogenase.

Essential characteristics of organisms to be utilized for microbialproduction of ethanol from plant biomass include the ability to secreteenzymes, transport glucose and xylose, metabolize glucose and xylose toethanol, as well as have sufficient ethanol tolerance to make thefermentation economically viable. It is unlikely an organism capable ofmeeting all of these criteria will be isolated from nature. Therefore,rational strategies to engineer strains for the industrial production ofethanol from plant biomass are preferred. The following characteristicsmake L. casei 12A an ideal Gram-positive species for research in thisarea:

-   -   Designation as a GRAS (generally Regarded As Safe) species.    -   Established platforms for introducing and expressing foreign        DNA.    -   Relatively simple fermentative metabolism with almost complete        separation of cellular processes for biosynthesis and energy        metabolism.    -   Resistance to environmental stress, including high        concentrations of acids and biofuels    -   Ability to use lignocellulosic carbohydrates.    -   Ability to secrete and display proteins, hence potential for use        in consolidated bioprocessing.

Multiple strategies were pursued concurrently to redirect L. casei 12Afermentation to ethanol. The first strategy involved inactivation ofenzymes that consume pyruvate under anaerobic conditions withoutproducing ethanol, including the D-Ldh; four L-Ldhs; D- (D-Hic);acetolactate synthase (Als); and oxaloacetate decarboxylase (Oad). Thisapproach has been used to inactivate L-ldh1, L-ldh2, and D-hic, as wellas to construct the L-ldh1/L-ldh2, double mutant. The highest level ofethanol formation was observed with the ΔL-ldh1/ΔL-ldh2 double mutant,which produces ethanol as 14% of its metabolic end products.

Our second strategy for increasing flux to ethanol involved expressingethanol producing enzymes. A codon optimized “PET” cassette comprised ofsynthetic Zymomonas mobilis genes encoding pyruvate decarboxylase (Pdc)and alcohol dehydrogenase (Adh2) was constructed, and placed under thecontrol of the L. casei 12A pgm promoter, pgm ribosomal binding site andkdgR transcriptional terminator. When this construct was introduced intoL. casei 12A, ethanol made up 61% of metabolic end products formed. Whenintroduced into L. casei 12A (ΔL-ldh1), ethanol was the dominant productobserved (91% of metabolic end productions). Results from this analysisindicate that the two approaches are complementary and demonstrate thatredirecting metabolic flux in L. casei from lactate to an alcohol can bereadily achieved. The organism described above was determine toaccumulate mannitol under high carbohydrate loading. Mannitolaccumulation was overcome by inactivation of the two mannitoldehydrogenase genes and the introduction of a second copy of the PETcassette under the control of the UspAC2 promoter. This derivative isdesignated Lb. casei E3.

The general strategy that was used to redirect metabolic flux in L.casei 12A from lactic acid to ethanol is illustrated in detail in FIG.3. Two different methods were used to carry out the strategy. The firstmethod, involving gene deletion, is illustrated in FIG. 4. The secondmethod, involving the construction and subsequent expression of asynthetic PET expression cassette construct in pTRKH2, is illustrated inFIG. 5. The growth of the resulting L. casei 12A ethanologens inChemically Defined Medium (CDM) is illustrated in FIG. 6. Thefermentation by-products of the L. casei mutants grown in CDM weremeasured, and the results are shown in Table 6.

TABLE 6 Fermentation products of L. casei 12A and mutants with andwithout pTRKH2 or pPGM-PET growth in CDM for 48 hrs. Ethanol L-LactateD-Lactate Derivative (%) (%) (%) 12A 0.0 95.0 5.0 12A ΔL-ldh1 6.0 49.045.0 12A ΔL-ldh2 0.0 96.0 4.0 12A ΔD-hic 0.0 71.0 29.0 12AΔL-ldh1ΔL-ldh2 14.0 34.0 52.0 12A(pTRKH2) 0.0 95.0 5.0 12A(Ppgm-PET)61.0 34.0 1.0 12A ΔL-ldh1(pTRKH2) 13.0 47.0 40.0 12A ΔL-ldh1(Ppgm-PET)90.9 1.5 1.5 Note: L. casei 12A mutants were grown in MRS from glycerolstock for 24 hrs at 37° C. then transferred to MRS and incubated for anadditional 18 hrs. CDM containing 50 mM glucose was inoculated andincubated in GC vials for 48 hrs at 37° C. At the 48-hr time point,supernanant was drawn off and submitted to GLBRC enabling technologiesfor fermentation by-product analysis via HPLC-RID.

Conclusions.

Inactivation of L-Ldh1 reduced flux towards L-lactate and enhanced fluxtowards D-lactate and ethanol. Inactivation of L-Ldh2 and L-Lhd3increased these changes in metabolic flux.

In L. casei 12A with the PET cassette, ethanol made up 61% of metabolicend products formed, while 91% of metabolic end productions weredirected to ethanol when the PET cassette was introduced into L. casei12A ΔL-ldh1.

The multi-pronged strategy, inactivating genes encoding enzymes thatproduce lactic acid or mannitol and introducing the PET cassette,effectively converted L. casei 12A from producing lactate as its mainmetabolic product to producing ethanol as its main metabolic endproduct.

REFERENCES

-   Cai, H., Thompson, R. L., Broadbent, J. R., and Steele, J. L.    (2009). Genome Sequence and Comparative Genome Analysis of    Lactobacillus casei: Insights into their niche-associated evolution.    Genome Biol. and Evol. 1:239-257.-   Duong, T., Miller, M. J., Barrangou, R., Azcarate-Peril, M. A., and    Klaenhammer, T. R. (2010). Construction of vectors for inducible and    constitutive gene expression in Lactobacillus. Microbiol Biotech,    4(3): 357-367.-   Kristich, C. J., Chandler, J. R., and Dunny, G. M. (2007).    Development of a host-genotype-independent counterselectable marker    and a high-frequency conjugative delivery system and their use in    genetic analysis of Enterococcus faecalis. Plasmid 57:131-144.

Example E. Use of an Alternate Promoter

In the previous examples, a first generation Lactobacillus caseiethanologen was created by a two pronged approach to redirect metabolicflux in L. casei 12A from lactate to ethanol. The first prong was toinactivate genes encoding lactate dehydrogenases, enzymes which competewith the 12A pathway to ethanol. The second prong was the introductionof synthetic genes modeled from Zymomonas mobilis that encode pyruvatedecarboxylase (Pdc) and alcohol dehydrogenase II (Adh2) activities (PETcassette). These genes were designed utilizing the L. casei codon usagefor highly expressed genes and placed under the control of L. caseiphosphoglycerate mutase promoter, thought to be a constitutivelyexpressed promoter.

This approach was highly successful, resulting in a strain that utilized504.5 mM glucose (9.1%) glucose in 96 h and produced 934.7 mM of“pyruvate-derived” metabolic end products, which is 92.6% of thetheoretical yield from 504.5 mM glucose in a 500 ml fermentation vesselunder anaerobic conditions at 37° C. in a defined media with 540 mMglucose. Ethanol was produced at a level of 771.3 mM (4.5%), which was82.5% of the metabolic end-products. The second most abundant metabolicend product was mannitol which was present at 110.1 mM after 96 h.

Mannitol accumulation began at approximately 21 h. At the same time,ethanol as a percentage of the total metabolic end products began todecrease (% ethanol in total), suggesting that pyruvate decarboxylaseactivity becomes limiting at that time. This corresponds to the entry ofthis organism into stationary phase, suggesting that the L. caseiphosphoglycerate mutase (pgm) promoter used to drive expression of thePET cassette is poorly expressed in stationary phase. Mannitolaccumulation was overcome by inactivation of the two mannitoldehydrogenase genes and the introduction of a second copy of the PETcassette under the control of the UspAC2 promoter. This derivative isdesignated Lb. casei E3.

REFERENCES

-   Lee, S. G., K. W. Lee, T. H. Park, J. Y. Park, N. S. Han, and J. H.    Kim. 2012. Proteomic analysis of proteins increased or reduced by    ethanol of Lactobacillus plantarum ST4 isolated from makgeolli,    traditional Korean rice wine. J. Microbiol. Biotechnol. 22:516-525.

Example F. Native Promoters with PET in Lactobacillus casei 12A ΔL-ldh1

To determine if the level and timing (growth phase) of expression L.casei pgm, groEL, dnaK and uspAC2 promoters impacted ethanol productionthese promoters were placed in front of the PET cassette and introducedinto L. casei 12A ΔL-ldh. These strains were grown in 5 ml MRS brothcontaining 2.5 μg/ml erythromycin (Erm) at 37° C. for 24 h. Then thecultures were transferred into 5 ml CHILL medium with 50 mM glucose and25 μg/ml Erm and continued to grow at 37 C for 24 h. The composition ofthese media is included as supplementary material. These cultures werewashed twice with saline solution (0.85% NaCl) and resuspended incomplete synthetic corn stover hydrolysate (SynH) containing 2.5 μg/mlErm. The OD₆₀₀ in all cultures was adjusted to 0.1 and then 1 mlaliquots of each culture were transferred into 2 ml GC vials. In eachvial, the septum on the cap was pierced by a needle (25G, 0.5 inches).This was done to prevent headspace pressure build up in the vial duringfermentation. Samples were incubated at 37° C. for 96 h. For each timepoint, cells were removed by centrifugation and the supernatants werefiltered through 0.2 μm syringe filters and subjected to HPCL analysisto identify metabolic end products. Two biological replications eachwith two technical replications were conducted.

The results of this analysis are presented in Table 7. These clearlydemonstrate that the pP_(uspA) results in the production ofsignificantly (p<0.05) more ethanol than any of the other promotersexamined. In stationary phase (hours 48 to 96) pP_(uspA) and pP_(pgm)produce 77.3 and 60.4 mM, respectively. This promoter was then utilizedto drive expression of a second copy of the PET cassette therebyreducing the accumulation of mannitol.

TABLE 7 Fermentation end products of L. casei 12A ΔL-ldh1 containingplasmids with the PET cassette under the control of different nativepromoters Ethanol produced in Time Fermentation end products, mM^(a)stationary Plasmid (h) Lactate Acetate Acetoin Ethanol phase*^(b)pP_(pgm)-PET 12  1.3 ± 0.4 4.3 ± 0.7  BQL^(c) 22.3 ± 0.1 60.4 24  6.2 ±0.8 4.2 ± 0.8 0.4 ± 0.3 68.0 ± 5.0 48 10.8 ± 0.3 5.5 ± 0.0 0.7 ± 0.1137.2 ± 1.7  72 13.9 ± 0.7 6.2 ± 0.5 0.7 ± 0.0 187.8 ± 4.1  96 14.9 ±1.6 5.6 ± 0.5 0.9 ± 0.0 197.6 ± 2.9  pP_(groEL)-PET 12  3.4 ± 0.1 4.6 ±0.1 BQL 15.8 ± 0.4 47.6 24 10.3 ± 0.1 2.0 ± 0.1 0.6 ± 0.0 46.7 ± 0.4 4827.4 ± 1.8 0.7 ± 0.7 1.1 ± 0.2 100.9 ± 4.7  72 36.3 ± 2.2 1.6 ± 1.1 1.3± 0.1 138.2 ± 9.0  96 38.2 ± 0.2 2.1 ± 1.5 1.7 ± 0.3 148.5 ± 3.9 pP_(dnaK)-PET 12  2.1 ± 0.5 2.8 ± 0.1 BQL  8.7 ± 2.7 24.7 24 10.6 ± 0.32.9 ± 0.0 0.9 ± 0.0 31.0 ± 0.3 48 31.6 ± 0.2 BQL 2.0 ± 0.2 78.0 ± 3.1 7238.5 ± 4.6 BQL 2.3 ± 1.3 98.5 ± 5.8 96 41.7 ± 6.6 BQL 2.4 ± 2.8 102.7 ±14.4 pP_(hypo)-PET 12  1.1 ± 0.5 2.1 ± 0.6 0.2 ± 0.6  9.1 ± 1.8 27.3 24 7.9 ± 1.3 4.6 ± 0.4 0.7 ± 0.4 23.9 ± 4.1 48 31.6 ± 1.0 BQL 2.7 ± 0.268.2 ± 3.6 72 44.4 ± 2.2 BQL 3.1 ± 0.5 88.0 ± 1.9 96 49.4 ± 1.5 BQL 3.2± 0.6 95.2 ± 3.6 pP_(uspA)-PET 12  4.7 ± 3.3 2.4 ± 1.3 BQL 14.1 ± 1.477.3 24 12.5 ± 0.2 5.9 ± 0.3 0.5 ± 0.0 54.6 ± 5.8 48 22.4 ± 1.0 5.7 ±0.5 0.6 ± 0.0 132.1 ± 2.5  72 30.3 ± 1.9 4.7 ± 2.3 0.7 ± 0.0 192.0 ±2.2  96 32.3 ± 3.0 3.9 ± 0.1 0.8 ± 0.0 209.4 ± 3.9  pTRKH2 12 3.50 ± 0.73.0 ± 1.1 BQL  6.1 ± 0.3 6.3 24  7.7 ± 0.9 4.4 ± 1.1 BQL  7.1 ± 2.1 4837.1 ± 6.0 BQL 2.4 ± 0.4 16.4 ± 4.2 72 64.4 ± 0.9 BQL 3.4 ± 0.0 24.7 ±1.3 96 72.7 ± 1.4 BQL 3.7 ± 0.3 22.6 ± 0.7 ^(a)Values are averages ±standard deviation. ^(b)Percent by w/v ^(c)BQL = Below quantitativelimit

Example G. The Use of Bacteriocin-Producing Lactic Acid BacteriaEthanologens to Control Contaminants in Biofuel Plants

Bacteriocins produced by LAB may display narrow or broad range of targetspecies. For example, nisin, a lantibiotic produced by some strains ofLactococcus lactis, and Class II bacteriocins such as pediocin andbrochocin, which are made by strains of pediococci, lactobacilli, andBrochothrix campestris, each display broad inhibitory activity against avariety of LAB spoilage microbes in alcoholic fermentations.

This example describes converting Lactobacillus casei 12A to anethanologen with the ability to express bacteriocins in lactic acidbacteria (LAB), including Lb. casei 12A. The bacteriocins produced bythe inventors' constructs allow the inventive ethanologens to inhibitcontaminating lactic acid bacteria (LAB) in ethanol plants, therebyreducing the production of lactate/acetate and enhancing ethanol yield.This methodology allows the reduction of lactate/acetate losses withoutthe use of antibiotics.

Experimental Approach and Results

Construction and Cloning of Genes Encoding Pediocin Production.

The sequence of the entire gene cluster required for the production ofpediocin (pedABCD) by Pediococcus acidilactici PAC 1.0 were obtainedfrom Genbank, codon optimized, and synthesized by GeneArt. These geneswere cloned into pNZ8048, a cloning vector demonstrated to be stablymaintained without antibiotic selection in Lactobacillus casei 12Aderivatives, under regulation by promoter Ppgm. The construct wasintroduced in Lb. casei E2 (Lb. casei 12A with its three primary lactatedehydrogenases (L-ldh1, L-ldh2, and D-hic) inactivated and whichcontains the production of ethanol cassette). This construct, designatedLb. casei E2 (pNZPed+) and the corresponding control strain Lb. casei E2(pNZ8048) were grown in MRS broth to stationary phase. Culturesupernatants were freeze-dried and resuspended in 10% of the originalvolume in sterile water. The concentrated supernatants from thesecultures and a pediocin control (Sigma) were mixed with Tricine samplebuffer with β-mercaptoethanol (Biorad) at 1:1 volume and heated at 95°C. for 5 min before loading into 10-20% Tricine SDS-PAGE (Biorad). Afterelectrophoresis, the gels were washed with sterile water overnight at 4°C., placed on MRS_(erm) agar and overlaid with 20 ml MRS_(erm) soft agarcontaining approximately 2×10⁶ CFU of Enterococcus faecalis CKIII. Theplates were in incubated at 37° C. until cell growth was detected. Theresults of this analysis are shown in FIG. 7. While the gel is slightlydistorted around the pediocin control, the pediocin-induced zone ofclearing from the Lb. casei E2 (pNZPed+) concentrated supernatant isreadily apparent.

Screening LAB Isolated from Ethanol Plants for Susceptibility toPediocin or Brochocin.

A variety of microorganisms are known to contaminate bioethanolfermentations, however LAB, particularly lactobacilli, are of primaryconcern (Beckner et al., 2011). The inventors' laboratory has isolatedsixty-two LAB from four ethanol plants in the Midwest region of theUnited States. This ethanol plant culture collection contains isolatesof Lb. fermentum, Lb. plantarum, Lb. brevis, Lb. mucosae, Lb.helveticus, Lb. heilongjiangensis, Lb. amylovorous, Lb. casei,Enterococcus sp. and Pediococcocus pentosaceus strains that weregathered from all stages of the ethanol fermentation process. Thirty ofthese cultures were propagated from frozen by sequential transfersthrough MRS broth and corn mash extract, a filtrate obtained from cornmash from a bioethanol plant. Pediocin (Sigma) was added at 500 ng/ml tocorn mash extract, inoculated with one of the ethanol plant isolates,incubated at 37° C. and growth was monitored by following O.D.₆₀₀.Growth of five of the thirty ethanol plant isolates was inhibited.

Construction and Cloning of Genes Encoding Brochocin Production.

The sequence of the genes required for brochocin production and immunity(brcABC) by Brochothrix campestris ATCC 43754 were obtained fromGenbank, codon optimized, and synthesized by GeneArt. These genes werecloned into pNZ8048, a cloning vector demonstrated to be stablymaintained without antibiotic selection in Lb. casei 12A derivatives,under regulation by promoter Ppgm and which included the pediocintransport genes pedCD. The brcABCpedCD construct was introduced in Lb.casei E2 (Lb. casei 12A with its three primary lactate dehydrogenases(L-ldh1, L-ldh2, and D-hic) inactivated and which contains theproduction of ethanol cassette. This construct, designated Lb. casei E2(pNZBrc+) and the corresponding control strain Lb. casei E2 (pNZ8048)were grown in MRS broth to stationary phase. Culture supernatants werecollected, freeze-dried and resuspended in 10% of the original volume insterile water. Agar diffusion assays of the crude BrcC preparationagainst LAB isolated from different ethanol plants showed 15 of 18ethanol plant isolates (which included strains of Lb. fermentum, Lb.plantarum, Lb. brevis, Lb. mucosae, Lb. helveticus, Lb. casei E2control, Enterococcus sp., and Pediococcocus pentosaceus) were sensitiveto this bacteriocin. Growth studies in a 96-well plate assay with 8representative ethanol plant isolates showed growth of all 8 strains inMRS medium was either completely (i.e., no growth after 48 h; Lb.fermentum A, Lb. mucosae 2C2, Lb. helveticus 3C2, Lb. plantarum 3C3, andLb. fermentum 3C9) or partially (i.e., growth observed after 48 h; Lb.brevis C, Lb. plantarum WC, and Pediococcus pentosaceus 4C0) inhibitedby BrcC.

Pediocin and brochocin are known to inhibit a particular range of LAB,and serve as exemplary model bacteriocins. Other bacteriocins with equalor even broader inhibition ranges may be, based on the presentdisclosure, synthesized and secreted in a similar manner. Table 8compares the effectiveness of three different LAB bacteriocins againstthe three antibiotics most commonly utilized in the ethanol industry aswell as another industry antimicrobial ingredient, hop acids. Theanalyses were conducted in corn steep liquor broth at 33° C. and pH 5.0,which represent conditions that mimic those present in bioethanolfermentations. Fifteen strains representative of the organisms isolatedfrom corn ethanol plants were utilized. Antibiotics and hop acids wereutilized at the concentrations suggested by companies selling theseproducts into the bioethanol industry. The bacteriocins were utilized ateither levels currently being produced (pediocin), at levels reported byothers in the research literature (nisin) using commercially availablepreparations, or in an assay system that allows for analyzing relativelybroad range of concentrations (brochocin inhibition assay by agardiffusion). Results demonstrate that brochocin and nisin have spectra ofinhibition that are similar to that for the antibiotics erythromycin andvirginiamycin. Additionally, these bacteriocins have an inhibitoryspectrum similar to that of hop acids in the yeast propagation tank andshow greatly superior inhibition versus hop acids in the fermentationvessels.

It is also known that bacteriocins often utilize the same transportsystems. Accordingly, the present methodology facilitates the generationof Lb. casei derivatives that secrete bacteriocins possessing a broaderspectrum of inhibition. Such inhibition reduces the production oflactate/acetate and enhancing ethanol yield in ethanol productionprocesses without the use of antibiotics.

TABLE 8 Sensitivity of 15 wild lactic acid bacteria isolated fromethanol plants to different inhibitors in corn steep liquor broth (CSL)at 33° C. and pH 5.5 Type Inhibitor Concentration Resistant SensitiveBacteriocins Pediocin 500 IU/mL^(a) 67% 33% Brochocin Agar Diff^(b)  9%91% Nisin 500 ng/mL^(c) 13% 87% Antibiotics Penicillin 6 μg/mL 33% 77%Virginiamycin 3 μg/mL 13% 87% Erythromycin 1 μg/mL  7% 93% Hop acids HopYPT^(d) 25 ppm 13% 87% Hop Ferm^(d) 1.5 ppm 87% 13% ^(a)Pediocin atconcentraiion produced by Lb. casei E3 (pNZPed+). ^(b)Assays done usingan agar diffusion assay using crude brochocin prep (freeze-driedsupernatant from overnight culture of Lb. casei E2 (pNZBrc+). Allows forevaluation of higher concentrations than CSL broth assays ^(c)Nisin atlevels attainable utilizing cultures of Lactococcus lactis. ^(d)Theconcentration of hop acids suggested by the manufacturer for use in theyeast propagation tank (YPT) or the concentration that would be presentin the fermenter after fill (Ferm).

A model system was developed to directly evaluate the ability ofengineered LAB such as Lb. casei E3 (pNZPed+) to inhibit yield reducingbacteria (YRB) in ethanol plants and increase ethanol yields. The modelutilizes corn mash prepared from industrial liquefact as thefermentation substrate and simulates the addition of a LABbacteriocin-producing ethanologen at the time an industrial fermenterstarts to be filled with mash. The liquefact is pasteurized by heatingin a water bath at 75° C. for 60 min prior to being utilized to reducethe level of contaminants in the corn mash. The experiment is started byadding a sufficient quantity of YRB to reach a level of 1×10⁵ CFU/g inthe corn mash (cooled liquefact), as this bacterial load is typical ofthat present in industrial corn mash. A quantity equal to 20% of thedesired final amount of corn mash containing YRB (i.e., 10 g) is addedto a 125 ml Erlenmeyer flask. Lb. casei E3 (pNZPed+) is added to theflask at a level that represents 1% of the final volume (i.e., 0.5 ml),resulting in the initial mash having approximately 5.0×10⁷ CFU/g of Lb.casei E3 (pNZPed+). The inoculated mash is incubated at 33° C.statically. Subsequently, additional 20% aliquots (i.e., 10 g) of thecontaminated mash (1×10⁵ CFU/g of the YRB) are made every 2 hours untilthe final volume (i.e., 50 g) is attained, 8 h after the start of theexperiment. The yeast strain such as “Ethanol Red” is added at hour 8 ata level of 1×10⁸ CFU/g, followed by addition of an enzyme mix(Distillase CS, DuPont Inc.) to reach 0.35 glucoamylase units/g DS ofcorn mash and sufficient urea to reach 500 ppm. Incubation is continuedat 33° C. for an additional 64 h (total time is 72 h) with mixing (150rpm for 3 min) every 24 h. The yeast addition is later than would occurin an industrial fermentation, however our preliminary results indicatethat the 8 hour incubation is required to observe consistent detectablelevels of lactate production. After 72 hours, samples are drawn for pHand HPLC analysis.

The pH at the end of the fermentation is an early indication of theability of a LAB bacteriocin-producing ethanologen such as LAB such asLb. casei E3 (pNZPed+) to inhibit YRB such as Lactobacillus strain LS1.As shown in FIG. 10, the yeast only fermentation resulted in a pH of4.93, and addition of the YRB strain LS1 results in a pH drop of 0.36units. Addition of a commonly used antibiotic (LacV) reduced but did noteliminate the drop in pH. In contrast, addition of Lb. casei E3(pNZPed+) to a yeast plus LS1 fermentation stopped the decline in pH.Addition of the non-bacteriocin producing Lb. casei E3 control (BacNegative) did not stop the YRB mediated pH decline. These resultsdemonstrate that bacteriocin production by E3 was effective in stoppingthe YRB mediated pH decline.

The decrease in pH that is observed with addition of YRB to the model isindicative of lactate and acetate production. The presence of theseorganic acids at the end of fermentation represents lost carbon thatcould instead have been converted to ethanol. Data for lactate levelsfrom the model trials are presented in FIG. 11. The yeast onlyfermentation contained 0.25% (w/v) lactate at the end of fermentation.Addition of the YRB LS1 resulted in an increase in lactate concentrationup to 0.62% (w/v) lactate. The addition of Lb. casei E3 (pNZPed+) to ayeast+LS1 fermentation resulted in a lactate concentration of 0.32%(w/v). Thus, Lb. casei E3 (pNZPed+) inhibited approximately 75% of thelactate production by LS1, and was nearly as effective as the antibioticLacV. This effect was not observed with the non-bacteriocin producingLb. casei E3 control (Bac Negative), which confirms that the bacteriocinproduced by Lb. casei E3 (pNZPed+) is responsible for inhibition oflactate production by LS1.

Carbon present in carbohydrates with a degree of polymerization 1 (DP1;i.e., glucose) and degree of polymerization 2 (DP2; i.e., maltose) holdssignificantly higher value as ethanol than in dried distiller grainswith solubles, yet are not converted to ethanol by yeast. Thus, thesugar represented by DP1 and DP2 “peaks” after fermentation representlost carbon that could be converted to ethanol by a LABbacteriocin-producing ethanologen such as LAB such as Lb. casei E3(pNZPed+). Data for residual DP1 and DP2 levels from the modelfermentations are presented in FIG. 12. The yeast only and yeast plusYRB (strain LS1) fermentations contained 0.95 and 0.89% (w/v) DP1/DP2 atthe end of fermentation, respectively. However, addition of Lb. casei E3(pNZPed+) or non-bacteriocin producing E3 (Bac Negative), to a yeast+LS1fermentation decreased DP1+DP2 levels to 0.70% or 0.65% (w/v) at the endof fermentation, respectively. Additionally, when Lb. casei E3 (pNZPed+)alone was added with the yeast, the DP1/DP2 level at the end offermentation fell to 0.56% (w/v). These results show Lb. casei E3derivatives consumed approximately 0.20% (w/v) of the carbohydrates inthe DP1 and DP2 “peaks” that are either not naturally or preferentiallyutilized by the yeast.

The primary objective of corn mash fermentations is the production ofethanol, and ethanol yield data from the model fermentations are shownin FIG. 13. The yeast only and yeast plus YRB (LS1) fermentationscontained 14.18 and 14.13% (w/v) ethanol at the end of fermentation,respectively; for an average of 14.16% (w/v) ethanol. Addition of Lb.casei E3 (pNZPed+) to either a yeast only or yeast+LS1 fermentationsresulted in 14.29 and 14.45% (w/v) ethanol, respectively, for an averageof 14.37% (w/v) ethanol. Thus, addition of Lb. casei E3 (pNZPed+)increased ethanol yield by approximately 0.2% (w/v). Collectively,results from the model fermentations (Figures A-D) demonstrate that aLAB bacteriocin-producing ethanologen such as Lb. casei E3 (pNZPed+) canenhance ethanol production efficiency by inhibiting bacterialcontaminants without the use of antibiotics, and by increasing the totalsugars that are converted to ethanol.

Example H. Heterologous Expression of Bacteriocins that InhibitYield-Reducing Lactic Acid Bacteria from Ethanol Plants

Rewiring L. casei 12A for ethanol production. To demonstrate thepotential of L. casei as a robust biocatalyst, we redirected metabolicflux in 12A from lactate to ethanol (Vinay-Lara at al. 2016). Briefly,multiple strategies were pursued concurrently to redirect L. casei 12Afermentation to ethanol; first, we inactivated enzymes that consumepyruvate under anaerobic conditions without producing ethanol,including: the D-lactate dehydrogenase (Ldh); four L-Ldhs;D-hydroxyisocaproate dehydrogenase (D-Hic); acetolactate synthase (Als);and oxaloacetate decarboxylase (Oad) using the pCJK47-based system werecently described [70]. 12A derivatives with altered metabolic endproduct profiles are presented in Table 8.

TABLE 8 Metabolic end products formed by L. casei 12A and derivativesduring growth in a chemically defined, glucose-media at 37° C., reportedas % of total. Derivative EtOH D-Lac L-Lac Ac Wild type 0.8 2.9 93.3 3.1ΔL-ldh1 9.2 54.6 36.2 BQL AD-Hic 0.6 0.1 96.3 3.1 ΔL-ldh1/ΔL-ldh2 5.487.5 5.5 1.5 ΔL-ldh1/ΔD-Hic 8.3 1.8 89.8 BQL ΔL-ldh1/ΔL-ldh2/ΔD-Hic 11.32.8 52.1 33.8 12A(pLc_P_(pgm)-PET) 10.7 9.2 78.5 1.6ΔL-ldh1(pLc_P_(pgm)-PET) 71.7 15.4 7.8 5.0 ΔL-ldh1/ΔL-ldh2/ΔD-Hic 90.20.8 3.1 5.9 (pLc-P_(pgm)-PET) ΔL-ldh1::P_(pgm)-PET[12A E1] 48.9 31.212.4 7.6 ΔL-ldh2/ΔD-Hic, 81.6 1.1 8.0 9.2 ΔL-ldh1::P_(pgm)-PET[12A E2]BQL = Below Quantifiable Level. Abbr: ETOH-ethanol, Lac-lactate,Ac-acetate.

The second strategy involved introduction of a L. casei codon-optimized“PET” cassette comprised of the Z. mobilis genes [71] for pyruvatedecarboxylase (Pdc) and alcohol dehydrogenase (Adh2) under the controlof the L. casei 12A phosphoglycerate mutase promoter (ppgm). Ppgm hasbeen reported to be a strong, constitutive in Lactobacillus species[72,73]. The PET cassette (Lc Ppgm-PET) was ligated into a copy numbervector, pTRKH2 [74], to produce pLc_Ppgm-PET, and introduced intovarious 12A derivatives. The metabolic end product profiles of key 12Aderivatives are presented in Table 8. Additionally, the Lc Ppgm-PETcassette was integrated into the 12A ΔL-ldh1 loci of 12AΔL-ldh1 and12AΔL-ldh1ΔL-ldh2ΔD-hic, these derivatives were designated 12AE1 and12AE2, respectively. The metabolic end product profiles of thesederivatives are also shown in Table 8. These results demonstrate: i) thetwo approaches are complementary; ii) redirecting metabolic flux in L.casei from lactate to an alcohol can be readily achieved; and iii) theplasmid-borne Lc Ppgm-PET cassette was more effective at redirectingmetabolic flux than the chromosomally integrated Lc Ppgm-PET cassette,suggesting gene dose is important.

The organism described above was determine to accumulate mannitol underhigh carbohydrate loading. Mannitol accumulation was overcome byinactivation of the two mannitol dehydrogenase genes and theintroduction of a second copy of the PET cassette under the control ofthe UspAC2 promoter. This derivative is designated Lb. casei E3.

Construction of a L. casei 12AE2 derivative capable of growth on xylose.The high level of xylose in plant biomass hydrolysates makes the abilityto utilize xylose (Xyl+) a priority in any biocatalyst to be used withthese feedstocks [75]. In most bacteria, xylose metabolism isaccomplished via conversion of xylose to D-xylulose and then to xylulose5-phosphate by xylose isomerase (XylA) and xylulose kinase (XylB),respectively [76,77]. In facultatively heterofermentative lactobacillilike Lb. casei, the phosphoketolase pathway is utilized to metabolizethe xylulose 5-phosphate to a mixture of lactate, ethanol, and acetate[78,79]. In LAB, it is also common for a xylose permease (XylT) to bepresent in xylose utilization operons [80,81]. To make L. casei 12AXyl+, we synthesized a codon-optimized 4,461 bp cassette encoding xylAand xylB from L. buchneri, and xylT from L. brevis behind the L. caseipgm promoter [82,83]. The xylose cassette was introduced into L. casei12A E2 either on pTRKH2 [74] or integrated into the chromosome in theL-ldh3 locus. The derivative containing the plasmid-borne cassette grewon xylose more rapidly and to a greater final cell density that the 12AE2 derivative with a chromosomal copy of the Xyl cassette, indicatingonce again that gene copy is important to Xyl+. As the result of removalof catabolite repressive elements, the 12A Xyl+ derivatives co-utilizeglucose and xylose and produce ethanol and acetate.

Redirecting flux from the phophoketolase pathway exclusively throughpyruvate. L. casei utilizes pentose sugars through the phosphoketolasepathway resulting in the formation of equimolar quantities of lactateand acetate [84]. Okano et al. [79] demonstrated it was possible toredirect pentose metabolism through the pentose phosphate pathway in LABby inactivating the phosphoketolase (xpk) gene via the introduction of aLactococcus lactis transketolase gene within its loci. A similarapproach has been applied to L. casei 12A E3 and determined to redirectxylose utilization through pyruvate; thereby allowing for higher yieldsof pyruvate-derived metabolites from pentoses.

Identification of constitutive promoters. A synthetic promoter librarywas developed in L. plantarum that spans 3-4 logs of expression in smallincrements [85]. This promoter library was also evaluated in L. sakeiand the level of expression correlated with that observed in L.plantarum [85]. To evaluate the activity of these promoters in L. casei,ppgm and three L. plantarum promoters with medium, medium-high, and highlevels of expression were inserted upstream of a gene encodingβ-glucosidase (gusA), ligated to pTRKH2 [74], and transformed into L.casei 12A E2. The three L. plantarum synthetic promoters resulted in theexpected medium, medium-high, and high levels of GUS activity, while thepgm promoter gave far lower GUS activity (˜10-fold lower than the lowestL. plantarum promoter). These results confirm the L. plantarum syntheticpromoter library will function in L. casei and suggest the pgm promoteris rather weakly expressed.

Inducible promoters. Metabolic engineering of LAB has been greatlyfacilitated by exploitation of an autoregulatory system for nisinproduction (NICE system), a bacteriocin produced by Lactococcus lactis[111], and the 3-component quorum sensing gene cluster for sakacin, abacteriocin produced by Lb. sakei [111]. Characterization ofsakacin-like bacteriocin clusters in FHL has demonstrated transcriptionof the bacteriocin structural gene is activated by interaction betweenan associated two-component signal transduction system (TCS) andautoinducing peptide (AIP) pheromone [112,113]. More importantly,vectors developed from the Lb. sakei AIP system have provided moretightly regulated and highly induced heterologous gene expression thanthe NICE system in two key FHL species, L. sakei and L. plantarum [114],and we have successfully deployed this system in L. casei. PlasmidpSIP411, which contains β-glucosidase (gusA) under the control of thesakacin inducible promoter [115], was electroporated into L. casei 12AE2. GusA expression in response to different levels of synthetic inducerpeptide (0-100 ng/ml) displayed a near linear dose response up to 50ng/ml, with a >300-fold dynamic range (FIG. 1).

Example I. Heterologous Expression of Bacteriocins and BacteriocinImmunity by a L. casei Ethanologen

Bioenergy production via fermentation of agricultural feedstocks isincreasing worldwide. Bioethanol remains the predominant end product,but commercial manufacture of next generation biofuels such asbiobutanol is also expanding. Irrespective of the product, bioenergyproduction from agricultural feedstocks is regularly subject tomicrobial contamination which negatively affects process efficiency andprofitability. Losses caused by microbial contamination at individual USbioethanol plants, for example, have been estimated at $14.5 million peryear (Muthaiyan et al. 2011), and there are over 200 bioethanol plantsoperating in the US. LAB are the most prominent group of contaminants(Beckner et al. 2011; Murphree et al. 2014; Steele and Broadbent,unpublished results), and cause both chronic and acute infections in theplant (Skinner and Leathers 2004). Chronic infections reduce ethanolyields by siphoning carbon away from yeast (Lucena et al. 2010;Muthaiyan et al. 2011), while acute infections curtail ethanolproduction by directly inhibiting yeast (Skinner and Leathers 2004).Addition of antibiotics, primarily penicillin and virginiamycin(Muthaiyan et al. 2011), is currently the most common means for controlagainst these loses. As expected, this practice has led to the emergenceof antibiotic-resistant contaminants (Murphree et al. 2014), and it mayalso affect the value of fermentation byproducts such as drieddistillers grains with solubles (Bischoff et al. 2016; McChesney 2009).Thus, process improvements that effectively control bacterialcontamination would substantially improve the production efficiency andcapacity of bioenergy production from agricultural feedstocks.

The four bacteriocins described in this prophetic example aresynthesized as pre-peptides with an N-terminal amino acid leadersequence, and secreted via a specific transporter encoded within thegene cluster responsible for bacteriocin production (Bierbaum and Sahl,2009; McCormick et al. 1998; Papagianni and Anastasiadou, 2009; vanBelkum et al. 2010) (FIG. 9). As is illustrated in FIG. 9, these geneclusters also include one or more genes that confer host immunityagainst the bacteriocin.

For this example, the inventors will create synthetic cassettes forexpression of each bacteriocin in L. casei E3, and separate cassettes toconfer immunity. The cassettes for immunity will be integrated into thechromosome of L. casei E3, while those encoding bacteriocin productionwill be carried on multi-copy plasmids. The coding regions in eachcassette will be codon optimized for L. casei using the Java CodonAdaptation Tool (JCAT, Technical University Braunschweig Institute forMicrobiology) with all annotated L. casei 12A genes as the input. As isnoted in the preceding examples, we have successfully expressedsynthetic pediocin A and brochocin-C gene clusters in L. casei E3; thisexample will focus on nisin and carnocyclin A.

The nisin production (NisP) cassette will include nisA-P genes describedin FIG. 9, and the nisin immunity (NisI) cassette will incorporatenisIEFG (Bierbaum and Sahl 2009). nisR and nisK genes will be excludedas they encode a two-component system for regulation of nisin production(Seizen et al. 1996) whose target sequence (the nisA promoter) will notbe included in the synthetic construct. Instead, the cassette will besynthesized with the dnaK promoter, which we have found is expressed ata moderate level during all phases of growth (Broadbent et al. 1997).

The NisI cassette will be assembled in a pCJK47 derivative, pBS1, andintegrated into the acetolactate decarboxylase (ald) locus of L. caseiE3. This plasmid is a suicide vector in L. casei and has been used forthe integration of constructs into the L. casei chromosome (Broadbent etal. 2014), and inactivation of ald has been determined to have no impacton growth in media not containing citrate. The L. casei NisI constructwill be verified by DNA sequence analysis and screened for nisinimmunity by the agar overlay method using Lactococcus lactis 11454 asthe nisin producer (Steele and McKay, 1986).

The NisP cassette is relatively large (˜9 kb), and so will besynthesized in four parts and assembled in L. casei NisI throughsequential cloning in pDW2. The final L. casei NisP::NisI construct willbe verified by DNA sequence analysis and screened for nisin productionand immunity by the agar overlay method using Lactococcus lactis LM0230as the nisin-sensitive indicator (Steele and McKay, 1986).

Synthesis of cassettes for production and immunity of the otherbacteriocins will be performed in similar fashion. The production ofcarnocyclin A (CclP) cassette will include cclBTCDA, and the immunitycassette (CclI) will encode cclIEFGH (van Belkum and Vederas 2012). Ineach case, the cassette design, use of the chromosomal ald site andplasmid DNA to insert genes encoding immunity and production,respectively, and confirmation of sequence and function will beperformed as described for NisP.

The quantity of bacteriocin in the cell-free culture supernatants(filter sterilized) will be determined using a serial dilution method.The cell-free culture supernatants will be diluted in 0.85% saline, 5 mmfilters will be saturated with the diluted supernatants, and then placedon MRS soft agar containing different LAB isolated from coren starchethanol plants. The inventors' laboratory has a bank of more than 150strains of LAB containing fifteen different species, with the following10 species being highest in abundance: Lb. helveticus, Lb. amylovorous,Lb. fermentum, Lb. brevis, Lb. casei, Lb. plantarum, Pediococcuspentosaceus, Lactococcus lactis, Weissella sp. and Enterococcus faecium.Screening will include one representative strain of the ten specieslisted above.

This example describes a series of L. casei strains that produce one offour different bacteriocins and are immune to all four. The presentstrategy will enable use of these strains in pairwise combinations thatleverage the different modes of action for each bacteriocin to reducethe selective pressure for resistance to any one bacteriocin andlengthen the time that these strains will be effective for the controlcontaminating bacteria in ethanol plants.

Each reference identified in the present application is hereinincorporated by reference in its entirety. While present inventiveconcepts have been described with reference to particular embodiments,those of ordinary skill in the art will appreciate that varioussubstitutions and/or other alterations may be made to the embodimentswithout departing from the spirit of present inventive concepts.Accordingly, the foregoing description is meant to be exemplary, anddoes not limit the scope of present inventive concepts.

We claim:
 1. An ethanologen for inhibiting contaminant lactic acidbacteria present in a biofuel manufacturing process, comprising anethanologen that is a lactic acid bacterium engineered to produce abiofuel and further engineered to produce a bacteriocin which inhibitscontaminant lactic acid bacteria present in the biofuel manufacturingprocess; wherein the ethanologen comprises: (a) one or more endogenousgenes encoding a mannitol dehydrogenase engineered to be inactive; (b)one or more exogenous genes encoding a pyruvate decarboxylase and one ormore exogenous genes encoding an alcohol dehydrogenase II; and (c) oneor more exogenous genes required for production of a bacteriocin;whereby the resulting engineered lactic acid bacterium produces morebiofuel than a wild-type lactic acid bacterium having the same geneticbackground in a biofuel manufacturing process and reduces lactate andacetate production in said process by secretion of the bacteriocin,which inhibits contaminant lactic acid bacteria.
 2. The ethanologen ofclaim 1, wherein the ethanologen is capable of fermenting sugars notnaturally or not preferentially utilized by a main fermenting microbepresent in the biofuel manufacturing process.
 3. The ethanologen ofclaim 2, wherein the main fermenting microbe is Saccharomycescerevisiae.
 4. The ethanologen of claim 1, wherein the lactic acidbacterium is Lactobacillus sp., Lactococcus sp., Enterococcus, sp. orStreptococcus sp.
 5. The ethanologen of claim 1, wherein the biofuel isethanol or isobutanol.
 6. The ethanologen of claim 1, wherein thebacteriocin is a Class I or Class II bacteriocin.
 7. The ethanologen ofclaim 1, wherein the bacteriocin is pediocin, nisin, brochocin-C, orcarnocyclin A.
 8. The ethanologen of claim 1, wherein the ethanologen isLactobacillus sp. engineered to produce the bacteriocin.
 9. Theethanologen of claim 8, wherein the Lactobacillus sp. is Lactobacilluscasei.
 10. The ethanologen of claim 1, wherein the exogenous genesrequired for production of the bacteriocin are operably-linked to aninducible promoter.
 11. The ethanologen of claim 1, further comprisingone or more immunity genes conferring resistance to the ethanologenagainst said bacteriocin.
 12. The ethanologen of claim 1, wherein theethanologen is for producing ethanol from one or more carbohydrates andthe carbohydrate is a lignocellulosic feedstock.
 13. The ethanologen ofclaim 1, wherein the biofuel manufacturing process is an antibiotic-freeprocess.
 14. A method of making an ethanologen, comprising: (a)inactivating within a lactic acid bacterium one or more endogenous genesencoding a mannitol dehydrogenase; (b) introducing into a lactic acidbacterium one or more exogenous genes encoding a pyruvate decarboxylaseand one or more exogenous genes encoding an alcohol dehydrogenase II;and (c) introducing into said lactic acid bacterium one or moreexogenous genes required for production of a bacteriocin; whereby theresulting engineered bacterium produces more biofuel than a wild-typelactic acid bacterium having the same genetic background in a biofuelmanufacturing process and reduces lactate and acetate production in saidprocess by secretion of the bacteriocin, which inhibits contaminantlactic acid bacteria.
 15. The method of claim 14, further comprising thesteps of: (e) inactivating within the lactic acid bacterium genesencoding proteins associated with catabolite repression or elementsrelated to catabolite repression; (f) removing DNA sequences encodingcatabolite responsive elements (cre) that are involved in repressing theexpression of genes required for uptake and metabolism of sugars notnaturally or not preferentially utilized by Saccharomyces cerevisiaeyeast; (g) introducing into the lactic acid bacterium one or moreexogenous genes required for uptake and metabolism of sugars notnaturally or not preferentially utilized by Saccharomyces cerevisiaeyeast; and (h) introducing into the lactic acid bacterium one or moregenes for hop- and/or antibiotic-resistance.
 16. An ethanologen for usein an ethanol manufacturing process, comprising a Lactobacillus casei12A derivative having: a deletion mutation ΔL-ldh1, an exogenous geneencoding a pyruvate decarboxylase, and an exogenous gene encoding analcohol dehydrogenase II, wherein the exogenous genes are operablylinked to a native L. casei promoter, wherein the engineered bacteriumproduces ethanol at a greater rate than a wild-type Lactobacillus casei12A bacterium having the same genetic background, and wherein theethanologen is capable of fermenting sugars not naturally or notpreferentially utilized by a main fermenting microbe present in theethanol manufacturing process wherein the ethanologen further comprisesone or more mannitol dehydrogenases engineered to be inactive wherebythe resulting engineered lactic acid bacterium produces more biofuelthan a wild-type lactic acid bacterium having the same geneticbackground in a biofuel manufacturing process and reduces lactate andacetate production in said process by secretion of the bacteriocin,which inhibits contaminant lactic acid bacteria.
 17. A method ofinhibiting contaminant lactic acid bacteria in a biofuel manufacturingprocess, comprising: (a) culturing an ethanologen of claim 1 on asubstrate comprising a carbohydrate; (b) inhibiting contaminant lacticacid bacteria present in the process by the ethanologen's secretion of abacteriocin; and (c) collecting a biofuel produced by the process.
 18. Amethod of reducing lactate and acetate production in a biofuelmanufacturing process, comprising: (a) culturing an ethanologen of claim1 on a substrate comprising a carbohydrate; (b) reducing lactate andacetate production by contaminant lactic acid bacteria present in theprocess by the ethanologen's secretion of a bacteriocin; and (c)collecting a biofuel produced by the process.
 19. The ethanologen ofclaim 1, wherein the ethanologen further comprises the following: (a)genes encoding proteins associated with catabolite repression orelements related to catabolite repression engineered to be inactive; (b)DNA sequences encoding catabolite responsive elements (cre) that areinvolved in repressing the expression of genes required for uptake andmetabolism of sugars not naturally or not preferentially utilized bySaccharomyces cerevisiae yeast engineered to be removed; (c) one or moreexogenous genes required for uptake and metabolism of sugars notnaturally or not preferentially utilized by Saccharomyces cerevisiaeyeast; and (d) one or more genes for hop- and/or antibiotic-resistance.